Composition of matter comprising liposomes embedded in a polymeric matrix and methods of using same

ABSTRACT

The present disclosure provides a composition of matter comprising liposomes encapsulating in their intraliposomal aqueous compartment at least one active agent, the liposomes having a diameter of at least 200 nm and being embedded in a water insoluble, water absorbed cross-linked polymeric matrix. In one embodiment, the composition of matter is held within an aqueous medium, preferably being in iso-osmotic equilibrium with the intraliposomal aqueous compartments of the liposomes. The present disclosure also provides a method of removal of non-encapsulated active agent from the composition of matter, a method of preparing said composition of matter, a pharmaceutical composition comprising said composition of matter, use of such composition of matter; a method of providing prolonged delivery of a active agent to a subject in need thereof by administering to said subject the composition of matter disclosed herein as well as a package comprising said composition of matter held within said aqueous medium and instructions for use thereof.

RELATED APPLICATIONS

This application is a national phase filing under 35 U.S.C. 371 ofInternational Application No. PCT/IL2009/000967, filed on Oct. 11, 2009,and claims the benefit of U.S. Provisional Application Ser. No.61/103,440, filed Oct. 7, 2008, the entirety of these applications arehereby incorporated herein by reference for the teachings therein.

FIELD OF THE INVENTION

The present disclosure is in the field of biochemistry and in particularto compositions of matter comprising a combination of polymers andliposomes for carrying active agents.

BACKGROUND OF THE INVENTION

Among other applications, liposomes are used as carriers of drugs fordelivery via a plurality of mechanisms. To this end, various types ofliposomes are used, from small unilamellar vesicles (SUV), largeunilamellar vesicles (LUV), multilamellar vesicles (MLV), multivesicularvesicles (MVV), large multivesicular vesicles (LMVV, also referred to,at times, by the term giant multivesicular vesicles, “GMV”),oligolamellar vesicles (OLV), and others. It is appreciated by thoseversed in the art that LMVV are somewhat different from unilamellarvesicles of various sizes and of the “onion like” MLV structure. In LMVVthe amount of aqueous medium forming the aqueous phase per the amount oflipid is greater than that in MLV, this potentially allowing higheramount of drug to be loaded into the aqueous phase, namely, higher drugto lipid mole ratio in the LMVV when compared to MLV system of similarsize distribution. This difference was exemplified by Grant et al. 2004[Anesthesiology 101(1):133-7, 2004] and in U.S. Pat. No. 6,162,462. Ithas been found that the difference in structure between MLV an LMVV notonly allows higher loading of the drug into the liposomes but also aprolonged release of the drug from the LMVV system.

Specifically, U.S. Pat. No. 6,162,462 discloses liposomal bupivacainecompositions in which the bupivacaine is loaded by a transmembraneammonium sulfate gradient, the liposomes being giant multivesicularvesicles (GMV, a synonym for LMVV) having a mole ratio of encapsulateddrug to lipid in said liposomal composition of at least 1.0. A specificdrug encapsulated in the liposomes of U.S. Pat. No. 6,162,462 is theamphipathic analgesic drug bupivacaine (BUP). These bupivacaine loadedLMVV have shown to be provide superior analgesia in mice and humans[Grant et al. 2004, ibid. and U.S. Pat. No. 6,162,462]. However aphenomenon that still remains unresolved with these LMVV relates toleakage of bupivacaine from the LMVV during storage at 4° C. or roomtemperature. Thus, after time, free drug is contained in the compositionof matter (the amount may be above drug MTD) and the administration ofthe composition of matter containing such free drug may result intoxicity and unwanted side effects (from exposure high amounts of freedrug), unfavorable pharmacokinetics and shorter duration of thetherapeutic effect. Thus, there is a need in the art to provide acomposition of matter where leakage of drug from liposomes encapsulatingsame during storage is reduced or prevented.

SUMMARY OF THE INVENTION

The present disclosure provides, in accordance with a first of itsaspects, a composition of matter comprising liposomes encapsulating intheir intraliposomal aqueous compartment at least one active agent, theliposomes having a diameter of at least 200 nm and being embedded in awater insoluble, water absorbed cross-linked polymeric matrix. In oneembodiment, this composition of matter is held in an aqueous medium,e.g. a storing medium. It has been found and shown herein that keepingthe composition of matter in a suitable aqueous medium, as furtherdefined herein, significantly reduces the amount of material that leaksfrom the liposomes.

Also provided by the present disclosure is a method of preparing acomposition of matter comprising liposomes encapsulating in theirintraliposomal aqueous compartment at least one active agent, theliposomes having a diameter of at least 200 nm and being embedded in across-linked water insoluble and water absorbed polymeric matrix, themethod comprising mixing (i) liposomes encapsulating in theirintraliposomal aqueous compartment at least one active agent, theliposomes having a diameter of at least 200 nm; (ii) at least onecross-linkable polymer; and (iii) an aqueous solution comprising across-linker capable of forming with said cross-linkable polymer a waterinsoluble, water absorbed cross-linked polymer having embedded thereinsaid liposomes.

Also provided herein is a method for removal of non-encapsulated activeagent from a composition of matter comprising liposomes encapsulating intheir intraliposomal aqueous compartment at least one such active agent,the liposomes having a diameter of at least 200 nm and being embedded ina cross-linked water insoluble and water absorbed polymeric matrix, thecomposition of matter being held in an aqueous medium, the methodcomprising decanting at least part of said aqueous medium from saidcomposition of matter, thereby removing from said composition of matterat least part of non-encapsulated active agent. This method isapplicable both for removing non-encapsulated material, e.g. followingpreparation, as well as removal of any material that has leaked from thecomposition of matter, e.g. during or following storage. As shownhereinbelow, after a period of three months storage leakage has beensignificantly reduced as compared to the same liposomes which have notbeen embedded in a polymeric matrix.

The present disclosure also provide a composition of matter prepared byany of the above recited methods. In one embodiment, once the aqueousmedium is decanted, the composition of matter comprises less than 10%non-encapsulated (free) active agent.

The present disclosure also provides pharmaceutical compositioncomprising as an active ingredient a composition of matter as disclosedherein as well as the use of the composition of matter as disclosedherein for the preparation of a pharmaceutical composition.

Finally, the present disclosure provides methods for providing prolongeddelivery of a active agent to a subject in need thereof, the methodcomprising administering to said subject said composition of matter or apharmaceutical composition comprising the same. Of particular interestit the method for providing prolonged analgesia. In this embodiment, theactive agent is an analgesic drug.

Finally, the present disclosure provides a package (or kit) comprisingat least one container comprising an aqueous medium holding acomposition of matter as disclosed herein or a pharmaceuticalcomposition comprising the same and instructions for decanting saidaqueous medium prior to use of said composition of matter to form, e.g.an administrable composition of matter.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carriedout in practice, embodiments will now be described, by way ofnon-limiting example only, with reference to the accompanying drawings,in which:

FIGS. 1A-1B are graphs showing the release of Bupivacaine (BUP), duringstorage at 4° C. (FIG. 1A) or at 37° C. (FIG. 1B), from largemultivesicular vesicles (LMVV) of different lipid compositions (BUP tophospholipid mole ratio of each is given) which have been loaded withBUP using remote loading driven by trans-membrane ammonium sulphate (AS)gradient.

FIGS. 2A-2B are graphs showing the release of Bupivacaine (BUP), duringstorage at 4° C. (FIG. 2A) or at 37° C. (FIG. 2B), from largemultivesicular vesicles (LMVV) of different lipid compositions (BUP tophospholipid mole ratio of each is given) which have been loaded withBUP using remote loading driven by trans-membrane calcium acetate (CA)gradient.

FIGS. 3A-3B are graphs showing the release of Bupivacaine (BUP), duringstorage at 4° C. (FIG. 3A) or at 37° C. (FIG. 3B), from LMVV ofdifferent lipid compositions (HSPC/CHOL 6/4 mole ratio; HSPC/C16SPM/CHOL3/3/4 mole ratio; and HSPC100/CHOL 6/4 mole ratio, BUP to phospholipidmole ratio of each composition is given) which have been loaded with BUPusing the passive loading approach.

FIGS. 4A-4B are graphs showing the temperature dependent % release ofBUP from alginate beads embedding LMVV (HSPC/C16SPM/CHOL 3/3/4 moleratio)-encapsulated BUP, where the LMVV have been loaded with BUP byremote loading using calcium acetate) gradient (CAgrad, FIG. 4A) orammonium sulfate gradient (ASgrad, FIG. 4B).

FIGS. 5A-5C are graphs showing the duration of analgesia in mice usingvarious liposomal systems identified in Table 8 as formulations 1 to 8(identified in the Figures with in the corresponding formulation number“x” as “lip x”), FIG. 5A showing the effect of injected volume ofliposomal BUP or in free form, the amount of BUP being constant 6mg/mouse; FIG. 5B showing the effect of 5 different LMVV formulations,the amount of BUP being constant 3 mg; and FIG. 5C which describes acomparison of the eight different LMVV formulations (Table 8) at a doseof 3 mg/mouse.

FIGS. 6A-6F are graphs comparing analgesia duration of two differentdoses of BUP (3 mg/mouse and 6 mg/mouse) for the five different LMVVformulations identified in Table 8 (“lip x” in FIGS. 6A-6E) and 2different amounts (0.375 and 0.75 mg/mouse) of non-encapsulated (free)BUP (in FIG. 6F); FIG. 6A comparing the effect of lip 2 (3 and 6 mgBUP/mouse), FIG. 6B comparing the effect of lip 3 (3 and 6 mgBUP/mouse), FIG. 6C comparing the effect of lip 4 (3 and 4.5 mgBUP/mouse), FIG. 6D comparing the effect of lip 5 (3 and 6 mg BUP), FIG.6E comparing the effect of lip 8 (3 and 6 mg BUP/mouse), and FIG. 6Fcomparing the effect of free (non liposomal) BUP at 0.375 mg/mouse usingtwo volumes (150 and 300 μl) and 0.75 mg/mouse at a volume of 150 μl.

FIG. 7 shows the kinetics of bupivacaine release at 37° C. from LMMVcomposed of HSPC100/C16SPM/CHOL (3/3/4 mole ratio) embedded in: alginatehydrogel (ALG) cross-linked with Ca⁺⁺ ions (referred to as ALG bupigel#3 and #4, as defined in Example 2 hereinbelow), ALG bupigelde-crosslinked with oxalic acid (ALG bupigel+OA, #5 as defined inExample 2 hereinbelow), chitosan (CHT) cross-linked with oxalate(referred to as CHT bupigel #6 or #7 as defined in Example 2hereinbelow) and CHT bupigel de-crosslinked with CaCl₂ (#8 CHTbupigel+CaCl₂ as defined in Example 2 hereinbelow).

FIG. 8 describes the in vivo analgesia duration achieved for LMVV(composed of HSPC100/C16SPM/CHOL having a 3/3/4 mole ratio) loaded withBUP via trans-membrane AS gradient and embedded in: alginate-Ca hydrogel(groups 2-5, as defined in Example 2 hereinbelow), chitosan-oxalate(groups 6-8, as defined in Example 2 hereinbelow); un-embedded Buploaded LMVV (group 1) and control group (group 9, as defined in Example2 hereinbelow) of free Bup.

FIGS. 9A-9L describe and compare the change in level of free Bup (% offree Bup in storage media) during storage at 4° C. of LMVV(HSPC100/C16SPM/CHOL having 3/3/4 mole ratio) loaded with Bup using AStrans-membrane gradient embedded in Ca-crosslinked alginate hydrogel(ALG-Beads) when stored in various storage media. FIGS. 9A-9B shows theaverage effect of storage (6 months) in saline, 0.2%, 0.5%, or 2.0% Bupsolutions (average of different batches of ALG-Beads produced accordingto the methods of the invention). FIG. 9C shows the average effect ofstorage (3 months) in saline 0.5%, 2.0% Bup solutions. FIGS. 9D to 9Fdescribe and compare the change in level of free Bup (% free Bup in thedifferent storage media) during storage at 4° C. of the differentbatches (herein denoted as A, B, C, D, E and an average plot) of LMVV(HSPC100/C16SPM/CHOL having 3/3/4 mole ratio) loaded with Bup using AStrans-membrane gradient embedded in Ca-crosslinked alginate hydrogelwhen stored in various storage media, during 3 months: saline (FIG. 9D),0.5% BUP (FIG. 9E) and 2.0% BUP (FIG. 9F). FIGS. 9G-9K describe andcompare the change in level of free Bup (% of free Bup in the differentstorage media) during storage at 4° C. of the different batches (hereindenoted as A, B, C, D, E and an average plot) LMVV (HSPC100/C16SPM/CHOLhaving 3/3/4 mole ratio) loaded with Bup using AS trans-membranegradient embedded in Ca-crosslinked alginate hydrogel when stored invarious storage media, during 3 months in: saline (FIG. 9G), in 0.2% BUP(FIG. 9H), in 0.5% BUP (FIG. 9I) or in 2.0% BUP (FIG. 9J). FIG. 9Kdescribes the average effect of storage (3 months) in saline, 0.2%,0.5%, or 2.0% Bup solutions (average of different batches of ALG-Beadsdescribed in FIG. 9G to 9J). FIG. 9L shows the average effect of storage(2 months) in saline 0.5%, 2.0% Bup solutions. FIGS. 9M-9Q describe andcompare the change in level of free bupivacaine (% of free Bup instorage medium) of LMVV (HSPC100/C16SPM/CHOL having 3/3/4 mole ratio)loaded with Bup via the AS trans-membrane not embedded in hydrogels whenstored in various storage media at 4° C. (FIGS. 9M, 9N and 9O in saline,0.5%, or 2.0% Bup solutions and FIG. 9P in saline, 0.2%, 0.5%, or 2.0%Bup solutions). FIG. 9Q describes a separate experiment of 2 monthsfollow-up upon storage in saline without hydrogel. All storage media inFIGS. 9A-9Q were brought to 285 mOsmole by addition of NaCl solution toretain iso-tonicity

FIG. 10A-10D describe the change in level of free Bup (% of free Bup instorage media) in different storage media of LMVV(HSPC100/C16SPM/CHOL3/3/4 mole ratio) loaded with Bup via trans-membraneAS gradient and embedded in Ca cross-linked alginate. Three storagemedia were used (saline, 0.5% Bup, and 2.0% Bup, all storage media werebrought to iso-tonicity of 285 mOsmole with NaCl solution). These werestored for 40 days at 4° C. and than used in the experiments describedin FIG. 10A-10D). FIGS. 10A and 10B describe release of Bup after theremoval of storage media by washing with saline followed by 30 hours ofincubation at 37° C. throughout this time amount of drug in the hydrogelmedia (saline) was measured (FIG. 10A) and also described as % Bupreleased (FIG. 10B). FIG. 10C describes the change in Bup level in thestorage media of the preparations incubated in their original storagemedia (0.5 and 2.0% Bup) at 37° C. for 25 hours. FIG. 10D is anextension of data from FIG. 10C to 15 days of incubation, FIG. 10E showsthe change in Bup concentration in 0.5% Bup, and 2.0% Bup storage mediaof the above described Bup loaded LMVV after incubation at a temperatureof 25° C. for 20 days.

FIGS. 11A-11E describe change in level of free Bup (% of free Bup instorage media) over a storage period of 3 months at 4° C., in differentliquid storage media of ALG-LMVV-BUP (HSPC100/C16SPM/CHOL 3/3/4 moleratio), remote loaded by trans-membrane AS gradient. The storage mediaused were: Saline (FIG. 11A), 0.2% BUP (FIG. 11B), 0.5% BUP (FIG. 11C),and 2.0% BUP (FIG. 11D). FIG. 11E summarizes the changes in Bupconcentration over a storage period of 3 months at 4° C., in differentliquid storage media of CHT-LMVV-BUP (HSPC100/C16SPM/CHOL 3/3/4 moleratio), remote loaded by trans-membrane AS gradient. The storage mediaused were: Saline, 0.2% BUP, 0.5% BUP, and 2.0% BUP.

FIG. 12 compares the duration of analgesia between ALG-LMVV-BUP beads(using LMVV of HSPC100/SPM/CHOL having mole ratio of 3/3/4), remoteloaded by AS gradient) stored in different liquid media (0.5%, 2%, 1%Bup solution, saline, or none (LMVV-BUP)), compared with blank ALG-Beads(without any encapsulated Bup), after 2 months storage at 4° C. and acontrol of 0.75% free BUP and administered after decantation of storagemedia.

DETAILED DESCRIPTION OF SOME NON-LIMITING EMBODIMENTS

The present invention is based on the understanding that existingbupivacaine liposomal formulations such as those described in U.S. Pat.No. 6,162,462 and by Grant et al. [Grant et al. 2004, ibid.] have atendency to leak drug during long term storage at low temperatures whichmay impose a risk of toxicity when administered to subjects in need ofthe drug. These bupivacaine liposomal formulations contained high drugto phospholipid (drug/PL) ratio (>0.5 mole/mole) in large multivesicularvesicle (LMVV, referred to in U.S. Pat. No. 6,162,462 as giantmultivesicular vesicles, GMV), albeit, following storage, a substantialamount of the a priori encapsulated drug was found to be in the externalmedium. Thus, a novel composition of matter was designed to allow theeasy removal of any non-encapsulated drug from a composition of matter,in case such leakage took place following storage of the composition ofmatter.

Specifically, it has been found that large liposomes, having a diameterof at least 200 nm (such as large multivesicular vesicles, LMVV) andbeing embedded in a cross-linked, water insoluble, water absorbedpolymeric matrix, can be stored in an aqueous medium; and followingstorage (e.g. just before use) any leaked drug (which is thus dissolvedin the aqueous storage medium), can be removed from the composition ofmatter by simple decanting (e.g. pouring, withdrawing, e.g. with apipette, or funnel) the aqueous storage medium from the composition ofmatter. Without being bound by theory, it is believed that due to thesize of the liposomes and their stable entrapment (capture) of theliposome loaded agent in the cross linked polymeric matrix, while thenon-encapsulated drug, freely dissolved in the storage aqueous mediummay be (almost fully) removed from the composition of matter once theaqueous medium, or at least a portion thereof, is withdrawn from thecomposition of matter.

Thus, in accordance with a first of its aspects, the present inventionprovides a composition of matter comprising liposomes encapsulating intheir intraliposomal aqueous compartment at least one active agent, theliposomes having a diameter of at least 200 nm and being embedded in awater insoluble, water absorbed cross-linked polymeric matrix. As willbe shown herein, the polymeric matrix is typically a hydrogel.

Two unique features are provided by the present invention. First, theembedment of liposomes in a hydrogel or hydrogel like matrix(cross-linked water absorbed and water insoluble polymeric matrix asdefined herein) reduces dramatically the leakage of active agent (e.g.drug) from the liposome and also prevents a change in the agent tophospholipid ratio in the liposomal formulation per se.

In one embodiment, the composition of matter is held in an aqueousmedium. Such composition of matter held in the aqueous medium (havingcharacteristics as defined herein) is suitable and ready for long termstorage, as will be further described below. It is important to notethat the amount of aqueous medium is such that when in a container, theaqueous medium preferably totally covers the water absorbed polymericmatrix, i.e. it is in excess compared to the water absorbed polymericmatrix. In fact, when viewing the container it appears to have twophases, one of the water absorbed polymeric matrix and the other of theaqueous medium covering it.

As used herein, the term “liposomes” denotes a system comprising anorganized collection of lipids forming at least one type of liposomes,and enclosing at least one intraliposomal aqueous compartment.

As used herein, the term “composition of matter” encompasses at leastthe combination of liposomes having a size of at least 200 nm and beingembedded in a matrix composed of at least one cross-linked (partially orfully) water immiscible, water absorbed polymer.

The liposomes within the composition of matter comprise at least oneliposome forming lipid, which forms the liposomes' membrane. Theliposomes' membrane is a bilayer membrane and may be prepared to includea variety of physiologically acceptable liposome forming lipids and, asfurther detailed below, non-liposome forming lipids (at the mole ratiowhich support the formation and maintenance of stable liposomes.

As used herein, the term “liposome forming lipids” is used to denoteprimarily glycerophospholipids and sphingomyelins which when dispersedin aqueous media by itself at a temperature above their solid ordered toliquid disordered phase transition temperature will form stableliposomes. The glycerophospholipids have a glycerol backbone wherein atleast one, preferably two, of the hydroxyl groups at the head group issubstituted by one or two of an acyl, alkyl or alkenyl chain, and thethird hydroxyl group is substituted by a phosphate (phosphatidic acid)or a phospho-estar such as phosphocholine group (as exemplified inphosphatidylcholine), being the polar head group of theglycerophospholipid or combination of any of the above, and/orderivatives of same and may contain a chemically reactive group (such asan amine, acid, ester, aldehyde or alcohol). The sphingomyelins consistsof a ceramide (N-acyl sphingosine) unit having a phosphocholine moietyattached to position 1 as the polar head group.

In the liposome forming lipids, which form the matrix of the liposomemembrane the acyl chain(s) are typically between 14 to about 24 carbonatoms in length, and have varying degrees of unsaturation or being fullysaturated being fully, partially or non-hydrogenated lipids. Further,the lipid matrix may be of natural source (e.g. naturally occurringphospholipids), semi-synthetic or fully synthetic lipid, as well aselectrically neutral, negatively or positively charged.

Examples of liposome forming glycerophospholipids include, without beinglimited thereto, glycerophospholipid. phosphatidylglycerols (PG)including dimyristoyl phosphatidylglycerol (DMPG); phosphatidylcholine(PC), including egg yolk phosphatidylcholine, dimyristoylphosphatidylcholine (DMPC), 1-palmitoyl-2-oleoylphosphatidyl choline(POPC), hydrogenated soy phosphatidylcholine (HSPC),distearoylphosphatidylcholine (DSPC); phosphatidic acid (PA),phosphatidylinositol (PI), phosphatidylserine (PS).

As appreciated, the liposome forming lipids may also include cationiclipids (monocationic or polycationic lipids). Cationic lipids typicallyconsist of a lipophilic moiety, such as a sterol or the same glycerolbackbone to which two acyl or two alkyl, or one acyl and one alkyl chaincontribute the hydrophobic region of the amphipathic molecule, to form alipid having an overall net positive charge. In cationic lipids, theheadgroup of the lipid carries the positive charge.

Monocationic lipids may include, for example,1,2-dimyristoyl-3-trimethylammonium propane (DMTAP)1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP);N-[1-(2,3-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammoniumbromide (DMRIE); N-[1-(2,3-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethyl-ammonium bromide (DORIE); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA);3β[N—(N′,N′-dimethylaminoethane) carbamoly] cholesterol (DC-Chol); anddimethyl-dioctadecylammonium (DDAB).

Polycationic lipids due to their large polycationic head group are notliposome forming lipids but rather they form micelle. However, whenmixed with other lipids such as cholesterol and various phospholipids atsuitable mole ratio the mixtures will form liposomes. The polycationiclipids include a similar lipophilic moiety as with the mono cationiclipids, to which polycationic head groups are covaletly attached such asthe polyalkyamines spermine or spermidine. The polycationic lipidsinclude, without being limited thereto,N-[2-[[2,5-bis[3-aminopropyl)amino]-1-oxopentyl]amino]ethyl]-N,N-dimethyl-2,3-bis[(1-oxo-9-octadecenyl)oxy]-1-propanaminium(DOSPA), and ceramide carbamoyl spermine (CCS). The cationic lipids mayform part of a derivatized phospholipids such as the neutral lipiddioleoylphosphatidyl ethanolamine (DOPE) derivatized with polylysine toform a cationic lipopolymer.

In one embodiment, the liposomes in the composition of matter compriseat least sphingomyelin. The term “sphingomyelin” or “SPM” as used hereindenotes any N-acetyl sphingosine conjugated to a phosphocholine group,the later forming the polar head group of the sphingomyelin (N-acylsphingosyl phosphocholines). The acyl chain bound to the primary aminogroup of the sphingosine (to form the ceramide) may be saturated orunsaturated, branched or unbranded. In one embodiment, the acyl chaincomprises between 12 to 24 carbon atoms (C12-C24), at times between 14to 20 carbon atoms. In some preferred embodiments, the SPM is a 06:0 orC18:0 sphingomyelin, namely, saturated C16 or C18 SPM. The SPM ispreferably a synthetic or semi-synthetic SPM, i.e. a derivative of anaturally occurring SPM and may include the D-erythro (2S,3R) as well asthe L-threo (2S,3S) isomers, although the former is preferable. Inaddition, in the context of the present disclosure, the sphingomyelin isalso the corresponding dihydro species in which, typically, although notexclusively, the trans double bond between C4 to C5 of the sphingosineis hydrogenated to form dihydrosphingosine), namely, anydihydrosphingomyelins (DHSM) corresponding to the SPM defined hereinabove. In yet a further embodiment, the mole ratio between the liposomeforming lipids other than SPM and said SPM is typically in the range of1:1 to 2:1, irrespective of the SPM used in accordance with the presentdisclosure.

Interestingly, it has been found that when the liposomes comprise intheir bilayer SPM at the amount of up to 75% of the total lipids formingthe liposome's bilayer, a decrease in the amount of drug leakage isobtained without compromising the rate of drug release from theliposomes at 37° C. and further without compromising the high loading ofthe drug into the liposomes. Thus, in one particular embodiment, thecomposition of matter comprises SPM content in the liposomes membrane inan amount between 25 to 75 mole % of the total phospholipids (liposomeforming lipids) in said membrane, or about 50 mole % of the total lipidswhen including cholesterol in the membrane.

Further, interestingly, it has been found and also shown herein belowthat the combination of hydrogenated soy phosphatidyl choline (HSPC)having a solid ordered (SO) to liquid disordered (LD) phase transition,characterized by a temperature T_(m) (a temperature in which the maximalchange in heat capacity occurs during the phase transition) of ˜53° C.,with C16SPM having its T_(m) at ˜41.4° C. led to the formation of astable composition of matter, i.e. reduced drug leakage during 4° C.storage, as compared to a composition of matter lacking C16SPM (whichwas less stable, namely, showing higher rate of drug leakage during 4°C. storage (i.e. same storing conditions)).

As mentioned above, the liposomes may also comprise other lipidstypically used in the formation of liposomes, e.g. for stabilization,for affecting surface charge, membrane fluidity and/or assist in theloading of the active agents into the liposomes. Examples of suchlipids, may include sterols such as cholesterol (CHOL), cholesterylhemisuccinate, cholesteryl sulfate, or any other derivatives ofcholesterol.

The liposomes may further comprise lipopolymers. The term “lipopolymer”is used herein to denote a lipid substance modified by inclusion in itspolar headgroup a hydrophilic polymer. The polymer headgroup of alipopolymer is typically water-soluble. Typically, the hydrophilicpolymer has a molecular weight equal or above 750 Da. Lipopolymers suchas those that may be employed according to the present disclosure areknown to be effective for forming long-circulating liposomes. There arenumerous polymers which may be attached to lipids to form suchlipopolymers, such as, without being limited thereto, polyethyleneglycol (PEG), polysialic acid, polylactic (also termed polylactide),polyglycolic acid (also termed polyglycolide), apolylactic-polyglycolicacid, polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline,polyaspartamide, polyhydroxypropyl methacrylamide, polymethacrylamide,poly dimethylacrylamide, polyvinylmethylether, polyhydroxyethylacrylate, derivatized celluloses such as hydroxymethylcellulose orhydroxyethylcellulose. The polymers may be employed as homopolymers oras block or random copolymers. The lipids derivatized into lipopolymersmay be neutral, negatively charged, as well as positively charged. Themost commonly used and commercially available lipids derivatized intolipopolymers are those based on phosphatidyl ethanolamine (PE), usually,distearoylphosphatidylethanolamine (DSPE).

One particular family of lipopolymers that may be employed according tothe present disclosure are the monomethylated PEG attached to DSPE (withdifferent lengths of PEG chains, in which the PEG polymer is linked tothe lipid via a carbamate linkage resulting in a negatively chargedlipopolymer, or the neutral methyl polyethyleneglycol distearoylglycerol(mPEG-DSG) and the neutral methyl poly ethyleneglycoloxycarbonyl-3-amino-1,2-propanediol distearoylester (mPEG-DS) [GarbuzenkoO. et al., Langmuir. 21:2560-2568 (2005)]. Another lipopolymer is thephosphatidic acid PEG (PA-PEG).

The PEG moiety has a molecular weight of the head group is from about750 Da to about 20,000 Da, at times, from about 750 Da to about 12,000Da and typically between about 1,000 Da to about 5,000 Da. One specificPEG-DSPE commonly employed in liposomes is that wherein PEG has amolecular weight of 2000 Da, designated herein ²⁰⁰⁰PEG-DSPE or^(2k)PEG-DSPE.

The liposomes are embedded in a polymeric matrix. The polymeric matrixcomprises at least one water insoluble, water absorbent polymer. Suchpolymeric matrix are known to form in an aqueous environment a hydrogel.The polymeric matrix is, preferably, biocompatible polymer. As usedherein, the term “matrix” denotes any network or network like structurethat may be formed from a fully cross-linked or partially cross-linkedpolymer. Thus, it is to be understood that hereinabove and below, whenreferring to a polymer, it also encompass more than one polymer formingthe matrix.

The cross-linked polymer forms a water insoluble (water immiscible)matrix. The term “water insoluble” is used to denote than upon contactwith water or a water containing fluid the cross-linked polymer(s) doesnot dissolve or disintegrates.

Further, in the context of the present disclosure, the cross-linkedpolymer forming the matrix is biocompatible, i.e. is inert to bodytissue, such that upon administration to a body, it will not be toxic,injurious, physiologically reactive or cause any immunological rejectionof the composition of matter.

The cross-linked polymeric matrix is also a water absorbing matrix andin the composition of matter in the context of the present disclosure isabsorbed with water. As used herein, the term “water absorbing” or“water absorbed” is used to denote that the polymer, once cross linked,is capable of absorbing water in an amount that is at least 4 times, attimes 10-50 times and even more of the polymer's or polymers' own weightthereby forming a gel. In the composition of matter of the presentdisclosure, the cross-linked polymeric matrix is soaked with waterthereby forming a hydrogel.

Water absorbing cross-linked polymers generally fall into three classes,namely, starch graft copolymers, cross-linked carboxymethylcellulosederivatives, and modified hydrophilic polyacrylates. Examples of suchabsorbent polymers are hydrolyzed starch-acrylonitrile graft copolymer;a neutralized starch-acrylic acid graft copolymer, a saponified acrylicacid ester-vinyl acetate copolymer, a hydrolyzed acrylonitrile copolymeror acrylamide copolymer, a modified cross-linked polyvinyl alcohol, aneutralized self-cross-linking polyacrylic acid, a cross-linkedpolyacrylate salt, carboxylated cellulose, and a neutralizedcross-linked isobutylene-maleic anhydride copolymer.

In one preferred embodiment, the matrix is a “hydrogel”. The term“hydrogel” as used herein has the meaning acceptable in the art.Generally, the term refers to a class of highly hydratable polymermaterials typically composed of hydrophilic polymer chains, which may benaturally occurring, synthetic or semi synthetic and crossed linked(fully or partially).

Synthetic polymers that are known to form hydrogels include, withoutbeing limited thereto, poly(ethylene oxide) (PEO), poly(vinyl alcohol)(PVA), poly(acrylic acid) (PAA), poly(propylene furmarate-co-ethyleneglycol) (P(PF-co-EG)), and polypeptides. Representative naturallyoccurring, hydrogel forming polymers include, without being limitedthereto, agarose, alginate, chitosan, collagen, fibrin, gelatin, andhyaluronic acid (HA). A subset of these hydrogels include PEO, PVA,P(PF-co-EG), alginate, hyaluronate (HA), chitosan, and collagen.

In one particular embodiment of the invention, the polymeric matrixcomprises alginate, such as, and at times preferably, low viscosity (LV)alginate (molecular weight of the polycarbohydrate ˜100,000), or verylow viscosity (VLV) alginate (molecular weight of the polycarbohydrate˜30,000). As also exemplified herein, the alginate was cross linked byCa ions to from Ca-alginate cross-linked hydrogel. The cross-linkedalginate is a water absorbing polymer, forming in the presence of watera hydrogel.

In one further embodiment, the polymer forming the matrix isbiodegradable. The term “biodegradable” refers to the degradation of thepolymer by one or more of hydrolysis, enzymatic cleavage, anddissolution. In this connection, when the matrix is a hydrogelcomprising synthetic polymer, degradation typically is based onhydrolysis of ester linkages, although not exclusively. As hydrolysistypically occurs at a constant rate in vivo and in vitro, thedegradation rate of hydrolytically labile gels (e.g. PEG-PLA copolymer)can be manipulated by the composition of the matrix. Synthetic linkageshave also been introduced into PEO to render it susceptible to enzymaticdegradation. The rate of enzymatic degradation typically depends both onthe number of cleavage sites in the polymer and the amount of availableenzymes in the environment. Ionic cross-linked alginate and chitosannormally undergoes de-crosslinking and dissolution but can also undergocontrolled hydrolysis after partial oxidization. The rate of dissolutionof ionic crosslinked alginate and chitosan depends on the ionicenvironment in which the matrix is placed. As will be illustrated belowby one embodiment it is possible to use cross-linked polymer and controlthe rate of degradation by addition at a desired time and a desiredamount of a de-crosslinker.

Specific examples of control of the cross-linking and de-crosslinkingmay include cross-linking the cationic chitosan with the di-carboxylicacid oxalate (OA) and de-cross-linking by the divalent cation calcium;and cross-linking the anionic alginate with the divalent cation calciumand de-crosslinking by either di-carboxylic acid such as oxalate (OA) orby chelating agents such as EDTA. Thus, at times, the composition ofmatter may be subjected to de cross linking.

The polymeric matrix may be present in the composition of matter in theform of individual particles, e.g. beads, each particle embeddingliposomes, or in the form of a continuous matrix. The particles may bespherical or asymmetrical particles, as appreciated by those versed inthe art of hydrogels.

The cross linked polymeric matrix are required to hold the liposomes. Asappreciated, liposomes in general may have various shapes and sizes. Theliposomes employed in the context of the present disclosure, may bemultilamellar large vesicles (MLV) or multivesiclular vesicles (MVV).

MVV liposomes are known to have the form of numerous concentric ornon-concentric, closely packed internal aqueous chambers separated by anetwork of lipid membranes and enclosed in a large lipid vesicle. In thecontext of the present disclosure, in order to be retained in thematrix, the liposomes have a diameter that is at least 200 nm. Thus, inone embodiment, the MVV are typically large multivesicular vesicles(LMVV), also known in the art by the term giant multivesicular vesicles(GMV). In accordance with one embodiment, the LMVV typically have adiameter in the range of about 200 nm and 25 μm, at times between about250 nm and 25 μm.

When the liposomes MVV, it is to be understood that the loading of theactive agent includes containment in more than one aqueous compartmentformed by the lipid membranes, and typically also in the aqueousenvironment surrounding the non-concentric.

The liposomes of the composition of matter encapsulate at least oneactive agent. Encapsulation includes the entrapment/enclosure, in theintraliposomal phase, of at least one active agent. The entrapment is anon-covalent entrapment, namely in the liposomal aqueous phase theactive agent is freely dispersed and may, under appropriate conditions,be released from the liposomes in a controlled manner.

The active agent may be a small molecular weight compound as well as apolymer (e.g. peptide, protein, nucleic acid sequence etc.). The term“active agent” is used to denote that the encapsulated agent, onceadministered has a beneficial effect, e.g. as a therapeutic, as acontrasting agent (e.g. radionuclei dyes or dye-conjugates to carrier,chromophor or fluorophor producing agent etc.), as a nutraceuticalcompound etc. The active agent may be a water soluble, hydrophiliccompound as well as an amphipathic compound.

In one embodiment, the active agent is an amphipathic compound. The term“amphipathic compound” is used to denote a compound possessing bothhydrophilic and lipophilic properties. There are various functionalamphipathic compounds known in the art. One example includes the anticancer compound doxorubicin. The loading of doxorubicin (e.g., DOXIL™)into preformed liposomes is driven by transmembrane ammonium sulfategradient (U.S. Pat. No. 5,192,549, U.S. Pat. No. 5,316,771 and Haran etal., [Haran G, et al. (1993) Transmembrane ammonium sulfate gradients inliposomes produce efficient and stable entrapment of amphipathic weakbases. Biochim Biophys Acta. 1151(2):201-15].

In one other embodiment, the amphipathic active agent is an analgesicdrug. The analgesic drug would typically be for local analgesic. Anon-limiting group of analgesic drugs are selected from the groupconsisting of benzocaine, chloroprocaine, cocaine, cyclomethycaine,dimethocaine, propoxycaine, procaine, proparacaine, tetracaine,articaine, bupivacaine, carticaine, cinchocaine, etidocaine,levobupivacaine, lidocaine, mepivacaine, piperocaine, prilocaine,ropivacaine, trimecaine, saxitoxin and tetrodotoxin. A preferred groupof analgesic drugs include, without being limited thereof, bupivacaine.lidocaine, ropivacaine, levobupivacaine, procaine, chloroprocaine,benzocaine, etidocaine, mepivacaine, prilocaine, ciprocaine, tetracaine,dibucaine, heptacaine, mesocaine, propanocaine, carbisocaine, andbutacaine. A specific analgesic drug according to the present disclosureis bupivacaine (hereinafter referred to, at times, as “BUP” or “bup”).

In another embodiment, the active agent is a water soluble molecule suchas water soluble steroid prodrugs, non steroidal anti inflammatory drug(NSAID), a peptide, protein or nucleic acid sequences, including, forexample, cytokines, antibodies, immunostimulating oligonucleotides(ISS-ODN), siRNA etc.

The composition of matter disclosed herein is characterized by a highagent to phospholipid (agent/PL) mole ratio, namely, high loading of theagent per liposome. Although not exclusively, the high loading wouldtypically depend on the type of liposomes used, their size, the loadingapproach etc. In one embodiment, a high loading is achieved by active(remote) loading approaches (see below) of the active agent into LMVV.In the context of the present disclosure, high loading is used to denotea loading with a active agent to lipid ratio in the resultingcomposition of matter of at least about 0.5 mole drug per mole liposomephospholipid (mole/mole) (this being characteristic of the LMVVaccording to the present disclosure).

Loading of the active agent into the liposomes may be by any techniqueknown in the art. Such techniques typically include passive loading oractive loading of agents into the liposomes.

Passive loading techniques of encapsulating agents into liposomestypically involve loading of the agent during preparation of theliposomes, e.g. by hydrating dry lipid mixture used to prepare liposomeswith a solution of the active agent. By passive loading the agent may beassociated to the liposomal membrane or encapsulated within theliposomes' aqueous core. One method for passive loading was described byBangham, et al., [Bangham A D, Standish M M, Watkins J C (1965)Diffusion of univalent ions across the lamellae of swollenphospholipids. J Mol Biol. 13(1):238-52], where an aqueous phasecontaining the agent of interest is put into contact with a film ofdried liposomes-forming lipids deposited on the walls of a reactionvessel. Upon agitation by mechanical means, swelling of the lipidsoccurs and multilamellar vesicles (MLV) encapsulating the agentdissolved in the hydration medium are thus formed. A further method forpassive loading is the Reverse Phase Evaporation (REV) method describedby Szoka and Papahadjopoulos, [Szoka F. C. Jr₅ Papahadjopoulos D. (1978)Procedure for preparation of liposomes with large internal aqueous spaceand high capture by reverse phase evaporation. Proc Natl. Acad. Sci.USA. 75(9):4194-8.], according to which a solution of lipids in a waterinsoluble organic solvent is emulsified in an aqueous carrier phase andthe organic solvent is subsequently removed under reduced pressure.Other methods of passive loading include subjecting liposomes tosuccessive dehydration and rehydration treatment, or freezing andthawing. Dehydration is carried out by evaporation or freeze-drying[Kirby C and Gregoriadis G (1984) Dehydration-Rehydration Vesicles: ASimple Method for High Yield Drug Entrapment in Liposomes. Nat.Biotechnol. 2, 979-984], or mixing liposomes prepared by ultra-sonicirradiation in aqueous solution with the solute to be encapsulated, andthe mixture is dried under nitrogen in a rotating flask. Uponrehydration, large liposomes are produced in which a significantfraction of the solute has been encapsulated [Shew R L, Deamer D W.(1985) A novel method for encapsulation of macromolecules in liposomes.Biochim Biophys Acta. 816(1):1-8]. Loading may be improvedco-lyophilizing the active agent with the dried liposome forming lipids[International Patent Application Publication No. WO03000227]

Active (remote) loading techniques are also used. For example, drugloading into pre-formed liposomes may achieved using a transmembrane iongradient or pH gradient. Loading using a pH and ion gradients may becarried out according to methods described in U.S. Pat. Nos. 5,616,341,5,736,155 and 5,785,987, U.S. Pat. No. 5,192,549, U.S. Pat. No.5,316,771 and Haran et al., [Haran G, et al. (1993) Transmembraneammonium sulfate gradients in liposomes produce efficient and stableentrapment of amphipathic weak bases. Biochim Biophys Acta.1151(2):201-15], incorporated herein by reference. The pH gradient maybe achieved using calcium citrate-based buffers having extra-liposomehigh pH and intra-liposome low pH. Intra-liposome high/extra-liposomelow ammonium sulphate-based gradient combines the ammonium ion gradientwith the pH gradient [Haran et al., 1993, ibid.].

In one embodiment, the composition of matter is held in an aqueousmedium. The aqueous medium is any water based buffer solution having adesired osmolality and ion composition and concentration and it is to beunderstood as encompassing a variety of physiologically acceptablebuffers. The buffer system is generally a mixture of a weak acid and asoluble salt thereof, e. g., sodium citrate/citric acid; or themonocation or dication salt of a dibasic acid, e. g., potassium hydrogentartrate; sodium hydrogen tartrate, phosphoric acid/potassium dihydrogenphosphate, and phosphoric acid/disodium hydrogen phosphate. A weak acidbuffer is a buffer solution with constant pH values of between 4 and 7and a weak base buffer is a buffer solution with constant pH valuesbetween 7 and 10. Some non-limiting examples of buffers that may be usedfor producing the aqueous medium in accordance with the presentdisclosure include physiological saline (0.9% NaCl), phosphate bufferedsaline (PBS), citrate phosphate buffer, sucrose histidine buffer,histidine buffer etc., set at a pH of between about 4 to 8, or between5.5 to 7 (as typically used in liposomal drug delivery systems).

In one embodiment, the aqueous medium is in iso-osmotic equilibrium withthe intraliposomal aqueous compartment of the liposomes within thepolymeric matrix. When referring to iso-osmotic equilibrium, it is to beunderstood as meaning that the aqueous medium and the medium of theintraliposomal aqueous compartment have similar osmolalities, thesimilarity being defined by a difference in osmolality of not more than50 mOsmole. In accordance with one embodiment, the osmolality of theaqueous medium and of the liposomal aqueous phase are in the range ofabout 50 to about RCE and Amendment in Response to Jul. 29, 2013 FinalOffice Action 600 mOsm/kg, or even between about 250 to about 550mOsm/kg. The iso-osmotic equilibrium may be obtained by holding thecomposition of matter comprising the polymeric matrix carrying theliposomes, with the buffer solution having an osmolality similar to thatof the intraliposomal aqueous compartment.

In one further embodiment, the equilibrium is achieved by using anaqueous medium comprising an amount of free active agent. The amount offree active agent is determined so as to form said iso-osmoticequilibrium. As shown in the examples herein, the presence of the freeagent (e.g. BUP in amounts of 0.2%, 0.5% or 2.0%) in the aqueous medium,reduced the leakage of the agent from the liposomes (this beingcomparable the same formulation without free drug in the aqueousmedium).

Interestingly, it has been found that the presence of free active agentin the aqueous medium not only reduced leakage from the liposomes, butalso increased the agent to phospholipid ratio in the liposomes (seeresults below).

As indicated above, the composition of matter may be in the form ofparticles, e.g. aggregates, beads, such as chitosan or alginate basedbeads, or any other type of discrete polymeric assemblies or maycomprise a continuous polymeric matrix. When the matrix comprisespolymeric particles, the latter may be dispersed or suspended in theaqueous medium.

The composition of matter is retained in the aqueous medium duringstorage. It has been envisages that even if during storage active agentleaks from the liposomes embedded in the matrix into the aqueous medium,upon need, e.g. before use, the storing aqueous medium may be decantedfrom the container holding the medium, thereby removing dissolved(non-encapsulated) agent from the composition of matter. Decanting theaqueous medium does not affect the liposomes entrapped within the matrix(entrapment being a result of the size of the liposomes). In otherwords, the encapsulated active agent are retained in place with theliposomes entrapped by the polymeric matrix (e.g. hydrogel). Thus, bysimple decanting or replacing the aqueous medium used during storage, itis possible to reduce risks of administering to a subject drug leakedfrom liposomes during storage.

In the context of the present disclosure, the stability of the liposomesmeans that the liposomes are chemically and physically unaltered whenstored at 4° C. and for a period of at least 3 months. The stability isdetermined, for example, by measuring the amount of active agent free inthe aqueous medium carrying the liposomes, i.e. non-encapsulated activeagent, the amount indicative of stability being less than 30%, 20% andat times even less than 10% from the total amount of active agent in thecomposition of matter (the total amount including encapsulated andnon-encapsulated agent).

While at minimum stable storage is for a period of 3 months, as will beshown in the following non-limiting examples, stable storage was alsoobtained for a period of four months (120 days), 4.5 months and even upto 6 months storing at 4° C. However, as indicated above, the stabilitywould be retained at any other temperature that is lower than thephysiological temperature of the body, namely, below 37° C. Whenreferring to lower temperatures it is to be understood that thereasonable storage temperature should be at least 15° C. below body coretemperature, i.e. below 22° C. According to one embodiment, storing isat a temperature between about 2° C. to 8° C.

The present invention also provides a method of preparing a compositionof matter comprising liposomes encapsulating in their intraliposomalaqueous compartment at least one active agent, the liposomes having adiameter of at least 200 nm and being embedded in a cross-linked waterinsoluble and water absorbed polymeric matrix, the method comprisingmixing (i) liposomes encapsulating in their intraliposomal aqueouscompartment at least one active agent, the liposomes having a diameterof at least 200 nm; (ii) at least one cross-linkable polymer; and (iii)an aqueous solution comprising a cross-linker capable of forming withsaid cross-linkable polymer a water insoluble, water absorbedcross-linked polymer having embedded therein said liposomes.

The cross linker in the context of the present disclosure is any agentcapable of cross linking polymers which as a result form a polymericmatrix that is water insoluble. The cross linker may form any type ofbond, including covalent bond, ionic bonds etc. with the polymer.Examples of cross linkers are provided hereinbelow. The selection of across linker will depend on the type of polymer used for forming thematrix, and the functional groups on the polymer. The cross-linker is atleast a bi-functional compound, at times a multifunctional compound,i.e. having two or more moieties capable of linking with two or morepolymers.

In accordance with this aspect, the method also comprises adding to saidcomposition of matter an aqueous medium having an osmolalitycorresponding to that of the intraliposomal aqueous compartment of saidliposomes (namely, the osmolality of the aqueous medium is adapted to beessentially the same as that in the liposomes). When referred to acorresponding osmolality it is meant that the aqueous medium has anosmolality that has no more than 50 mOsmole with the osmolality of theintraliposomal aqueous core of the liposomes within the matrix. Theosmolality in the intraliposomal aqueous core may be pre-established bythe conditions at which the liposomes encapsulating the agent areformed, or it may be determined by techniques known in the art, allbeing in line with the biochemists general knowledge in liposometechnology. The amount of aqueous medium typically added to thecompetition of matter is such that the composition of matter is entirelycontained in the medium.

Also provided by the present disclosure is a method for removal ofnon-encapsulated active agent from a composition of matter comprisingliposomes encapsulating in their intraliposomal aqueous compartment atleast one such active agent, the liposomes having a diameter of at least200 nm and being embedded in a cross-linked water insoluble and waterabsorbed polymeric matrix, the composition of matter being held in anaqueous medium, the method comprising decanting at least part of saidaqueous medium from said composition of matter, thereby removing fromsaid composition of matter at least part of non-encapsulated activeagent.

The aqueous medium may be partially withdrawn or substantially fullyremoved, to provide a composition of matter essentially free ofnon-encapsulated active agent. Essentially free of active agent denotesthat the resulting composition of matter comprises at most 10%, attimes, 7% of any non-encapsulated active agent (free active agent). Itis noted that even if all aqueous medium is removed from the system, dueto the characteristics of the cross-linked polymeric matrix, namely,being absorbed with a high content of water, the compression of matterremains in a hydrogel form.

As may be appreciated by those reading the current disclosure, the aboveremoval method is of particular advantage for compositions of matterafter long term storage. As discussed above, during long term storage(several months and more) liposome encapsulated agents tend to leak fromthe liposomes into the surrounding medium. Thus, once the liposomes areentrapped in a polymeric matrix held within a medium where thenon-encapsulated agent (e.g. drug) is dissolved, the removal of theaqueous medium with the dissolved agent allows to reduce the riskinvolved in administering free active agent.

The present disclosure also provides a pharmaceutical compositioncomprising as an active ingredient the composition of matter as hereindefined, or as obtained by the method disclosed herein. For instance,the present disclosure provides a pharmaceutical composition prepared bythe removal method disclosed herein.

The present invention also provides the use of the composition of matteras defined hereinabove for the preparation of a pharmaceutical ordiagnostic composition, for, respectively, treatment of a medicalcondition or for diagnostic purposes. The composition typicallycomprises, in addition to said composition of matter, at least onephysiologically acceptable additive.

Further, the present invention provides a method for the treatment ordiagnostic of a medical condition, the method comprising administeringto a subject in need of said treatment or diagnostic an amount of thecomposition of matter as defined hereinabove or in combination with atleast one physiologically acceptable additive comprising the same.

Without being bound by theory, it is believed that the administration ofthe liposomes embedded in the polymeric matrix would reduce uptake ofthe liposomes by macrophages, thereby increase the retention time of thecomposition of matter at the site of administration.

The composition of matter, alone (i.e. as is after removal of aqueousmedium) or in combination with physiologically acceptable additives maybe administered by any route acceptable in the art. According to oneembodiment, the administration of the composition of matter is byparenteral injection or infusion. This would include, without beinglimited thereto, intravenous, intraarterial, intramuscular,intracerebral, intracerebroventricular, intracardiac, subcutaneous,intraosseous (into the bone marrow), intradermal, intratheacal,intraperitoneal, intravesical, and intracavernosal and epiduaral(peridural) injection or infusion. Pareneral administration may alsoinclude transdermal, e.g. by transdermal patches, transmucosal (e.g. bydiffusion or injection into the peritoneum), inhalation and intravitreal(through the eye).

In one embodiment, the administration is by direct deposition of thecomposition of matter at the target site, e.g. by injection using asyringe, or by dip-coating an implant to be placed at a target site. Thepolymeric composition of matter may be placed at a site of injury suchas bone, teeth, muscle, tendon, heart, etc.

One particular embodiment of the invention comprises administration of acomposition of matter as defined herein to the bone or near a tooth totreat with an anti-bacterial, anti-fungal and/or anti-viral agent, incombination with growth factors so as to support both or teethregeneration. Treatment of osteoarthritis is one particular embodimentof the present disclosure.

When the active agent is an analgesic drug, a preferred mode ofadministration is local administration at a desired site by anyacceptable route, as can be determined by a medical doctor or any otherappropriate physician. With respect to analgesia, the desired site maybe, without being limited thereto, bone tendon, joints, GI tract,breast, prostate, reproductive tract, cardiovascular system or anyinternal organ such as the liver, kidneys, spleen, pancreas etc.

Further, when the active agent is an analgesic drug, the composition ofmatter may be used for local pain management and/or for treatment oflocal inflammatory reactions, optionally in combination with othertherapies. A non-limiting application may include treatment of arthritise.g. osteoarthritis or rheumatoid arthritis.

Further, in the context of the present disclosure, the desired site mayalso serve as a depot and as a reservoir for prolonged systemic release,such as for contraceptive, antibiotics, antiviral anti fungal antiparasite agents, immunosuppressants, hormones and growth factors,neurotransmitors, etc. and in such a case the administration route maybe subcutaneous.

The amount of composition of matter administered, and thereby the amountof active agent encapsulated therein should be effective to achieve thedesired effect by the active agent, at the target site. For example, ifthe active agent is a drug, the amount of the composition of mattersshould be determined so that at the target site the amount of the drugencapsulated therein is sufficient to achieve the desired therapeuticeffect. Such desired therapeutic effect may include, without beinglimited thereto, amelioration of symptoms associated with a medicalcondition, prevention of the manifestation of symptoms associated with amedical condition, slow down of a progression state of a medicalcondition, enhance of onset of a remission period, prevent or slow downirreversible damage caused by the medical condition, lessen the severityof the medical condition, cure the medical condition or prevent it fromdeveloping, etc. The medical condition to be treated by the compositionof matter may be any such condition treatable by the active agentencapsulated in the liposomes according to the present disclosure.

Further, if the active agent may be a diagnostic agent. To this end, theamount of the composition of matter should be such that it would bepossible to image the marker at the target site.

The amount of the composition of matters will be determined by suchconsiderations as may be known in the art, typically using appropriatelydesigned clinical trials (dose range studies etc.).

Finally, the present disclosure provides a package (kit) comprising atleast one container (e.g. syringe, vial) comprising an aqueous mediumholding a composition of matter or a pharmaceutical composition asdefined herein, and instructions for decanting said aqueous medium priorto use of said composition of matter. The instruction may include simplemechanical pouring of the aqueous medium, or when the composition ofmatter and aqueous medium are held in a syringe, the removal of theaqueous medium may include simple pressing out the aqueous medium fromthe syringe (having a lumen too thin to pull out therefrom thehydrogel).

As used herein, the forms “a”, “an” and “the” include singular as wellas plural references unless the context clearly dictates otherwise. Forexample, the term “a liposome forming lipid” includes one but also morelipids capable of forming by themselves a liposome when dispersed inaqueous medium.

Further, as used herein, the term “comprising” is intended to mean thatthe composition of matter include the recited constituents, i.e. theliposome forming lipid, and the active agent, but not excluding otherelements, such as physiologically acceptable carriers and excipients aswell as other active agents. The term “consisting essentially of” isused to define composition of matters which include the recited elementsbut exclude other elements that may have an essential significance onthe effect to be achieved by the composition of matter. “Consisting of”shall thus mean excluding more than trace amounts of other elements.Embodiments defined by each of these transition terms are within thescope of this invention.

Further, all numerical values, e.g. when referring the amounts or rangesof the elements constituting the composition of matter comprising theelements recited, are approximations which are varied (+) or (−) by upto 20%, at times by up to 10% of from the stated values. It is to beunderstood, even if not always explicitly stated that all numericaldesignations are preceded by the term “about”.

The invention will now be exemplified in the following description ofexperiments that were carried out in accordance with the invention. Itis to be understood that these examples are intended to be in the natureof illustration rather than of limitation. Obviously, many modificationsand variations of these examples are possible in light of the aboveteaching. It is therefore, to be understood that within the scope of theappended claims, the invention may be practiced otherwise, in a myriadof possible ways, than as specifically described hereinbelow.

DESCRIPTION OF SOME NON-LIMITING EXAMPLES

Materials

Drugs:

Bupivacaine hydrochloride (BUP) was supplied by Sigma St. Louis, Mo.

Methylprednisolone sodium succinate (MPS) was supplied by PHARMACIANV/SA Puurs-Belgium.

Lipids:

Cholesterol (CHOL) was supplied by Sigma, St. Louis, Mo. (better than99% pure, standard for chromatography grade).

Fully hydrogenated soy phosphatidylcholine (hereinafter “HSPC-100” or“H100”), Phospholipon® 100H batch no 50190 obtained from PhospholipidsGmbH Nattermannallee l*D 50829 Koln, Germany.

Fully hydrogenated soy phosphatidylcholine (hereinafter “HSPC”) wasobtained from Lipoid Gmbh, Ludwigshafen, Germany.

Fully synthetic N-Palmitoyl-D-erythro-sphingosine-1-phosphocholineN-palmitoyl sphingomyelin, (hereinafter “C16SPM”) >98% pure (lot no546701) was obtained from Biolab Ltd., POB 34038 Jerusalem 91340.

Buffers and Salts:

Ammonium sulfate (AS) was obtained from MERCK.

Calcium acetate monohydrate (CA) was obtained from Aldrich.

Calcium chloride-dihydrate obtained from MERCK.

EDTA obtained from Sigma.

Water Insoluble, Aqueous Absorbent Polymers:

Alginic acid sodium salt (Alginate=ALG), from brown algae, viscosity of2% solution at 25 c:250 cps LV-ALG (low viscosity) from SIGMA.

Sodium alginate pronova up VLVG-ALG (very low viscosity grade) batch#BP-0709-03 from NOVAMATRIX Sandvika, Norway, Was obtained from Sigma.

Preparation of Drug Loaded LMVV:

Preparation of Large Multi Vesicular Vesicles (LMVV)

Powder mixtures of lipids at the desired mole ratio, as specified in thevarious experiments hereinbelow, were dissolved in ethanol at 60-65° C.and added to an aqueous solution of ammonium sulfate (AS), calciumacetate (CA) or another buffer (as indicated below) to reach a finalphospholipid (PL) concentration of 60 mM and final ethanol concentrationof 10%.

The resulting solutions were mixed for 30 min at 65° C. to obtainmultilamellar large vesicles (MLV). Alternative methods to prepare MLVcan also be used (see for example: Barenholz & Crommelin, 1994, In:Encyclopedia of Pharmaceutical Technology. (Swarbrick, J. and Boylan, J.C., Eds.), Vol. 9, Marcel Dekker, N.Y. pp. 1-39).

LMVV were prepared from the MLV with the desired aqueous phase (forexample: ammonium sulfate 250 mM or 127 mM, calcium acetate 250 mM, or200 mM, or a desired buffer) from the MLV by exposing the MLV to 10cycles of freezing in liquid nitrogen and thawing in a water bath at 60°C. thereby forming the LMVV. At each cycle, for each 1 ml of dispersedLMVV solution the frozen dispersion was kept at the liquid nitrogen for1 minute. For example, a dispersion of 3 ml was kept in liquid nitrogenfor 3 minutes.

Gradient Creation

Transmembrane AS or CA gradient were created by removal of AS or CA(respectively) from the extra liposome aqueous phase and replacing itwith NaCl.

Three methods were used for creating the pH gradient:

(i) Centrifugation (Grant et al 2004, ibid.] for both AS and CAgradients at 1000 g, for 5 min at 4° C. Supernatant was removed andpellet was washed with saline at 4° C. The washing process was repeated7 times.

(ii) Dialysis using MWCO 12-14000 Dalton dialysis tubing.

(iii) Diafiltrating using Midjet benchtop system with hollow fibercartridge 500000 NMWC (GE Healthcare Bio-Sciences Corp. Westborough,Mass. 01581 USA).

Loading of Bupivacaine

LMVV were loaded with Bupivacaine (BUP) using two alternativeapproaches:

(i) Remote loading of preformed liposomes having a trans-membraneammonium sulfate (AS) gradient (Haran et al., (1993), BBA, 1151201-215), modified to fit the LMVV (Grant et al 2004, ibid.); Like inthe remote loading od doxorubicin, this approach take advantage of thefact that BUP is an amphiphatic weak base; or remote loading intopreformed LMVV having a trans-membrane calcium acetate (CA) gradient(Clerc & Barenholz. (1995), BBA, 1240, 65-257, Avnir et al (2008)Arthritis & Rheumatism, 58, 119-129). This method makes use of the factthat BUP, due to its tertiary amine is also an amphipathic weak acid

(ii) Passive loading was performed by lipid hydration using aqueoussolutions of BUP to form the BUP loaded MLV from which BUP loaded LMVVwere prepared as described above (LMVV preparation).

In both approaches loading was performed at 60-65° C., which is abovethe HSPC and C16SPM solid-ordered (SO) to liquid-disordered (LD) phasetransition temperature range (characterized by the relevant T_(m)). Itis noted that HSPC and C16SPM are the liposome-forming lipids of theLMVV described here.

Remote loading was performed for 30 min. at 60-65° C. using 4.5%, 5.5%,or 5.7% BUP, which is equivalent to osmolarity of (saline=0.9% weightper volume), or 6% BUP in distilled water as the liposome externalaqueous phase. An amount 0.5 ml of a wet LMVV pellet and 2 ml of BUPsolution were used for the remote loading. The mixture was then cooledto 4° C. in which it was stored overnight followed by unloaded free drugremoval (as described below).

Passive loading of BUP was performed by hydrating the ethanol lipidsolution with aqueous solution of distilled water containing 4.5% (231mOsm/kg), or 5.5% (285 mOsm/kg), or 6% (301 mOsm/kg) or 7% (346mOsm/kg), or 8% (373 mOsm/kg) or 10% (454 mOsm/kg) BUP (W/V) at 65° C.for 30 min. For this process 0.5 ml ethanolic lipids solution containing225 mg phospholipids and 77 mg CHOL were used. This solution was mixedwith 5 ml of one of the above indicated BUP aqueous solutions. Thesuspension was processed by 10 repetitive freezing and thawing cycles(as described above) and than kept overnight in a cold room (4°-6° C.)followed by free drug removal (as described below).

Free Drug Removal

Non-encapsulated BUP was removed from LMVV by washing with saline (1 mlliposomes/4 ml saline) and centrifuging the dispersion at 1000 g for 5min at 4-5° C. The washing process was repeated 7 times. The finalmedium (referred to herein as the “aqueous medium”) used to replaceextra-liposome from CA gradient loaded liposomes was PBS. The use of PBSwas preferred over saline. AS and the medium used for passive loading ofliposomes was replaced and LMVV were washed with un-buffered saline.

The LMVV was concentrated to a final solution of 2% BUP for the passiveloading and AS gradient loading. For CA gradient loading LMVV with 1%BUP final concentration was used, due to the large volume of these LMVV.These concentrations were close to the highest concentrations used formice injection with BUP in LMVV

The stability of LMVV thus formed was measured with respect to therelease rate of BUP from liposomes during storage at 4° C.

Bupivacaine Loading Under Iso-Osmotic Conditions

When referring to iso-osmotic conditions, it should be understood tomean that the osmolality of the intraliposomal aqueous core and theexternal medium inside and outside the liposomes are essentiallyidentical or close, all as defined hereinabove.

Three osmomolar concentrations were tested:

(i) 280 mOsm/kg isoosmotic to physiological saline (0.9% NaCl)condition: the AS and CA gradient LMVV were prepared with ˜20 mg/ml ASor CA solution adjusted by AS or CA solutions to 280 mOsm/kg. BUPloading concentration was 5.7% BUP in water or 4.5% BUP in NaCl solutionto reach 280 mOsm/kg.

(ii) 550 mOsm/kg, isoosmotic to 250 mM AS: the washing solution forcreating the AS gradient and the solution for removal of the free drugafter loading was NaCl solution. adjusted to 550 mOsm/kg. The drugloading conc. was 4.5% BUP in NaCl solution, or 4.5% BUP in sucrose sol.to make 550 mOsm/kg.

(iii) 650 mOs, isoosmotic to 250 mM CA.

Methyl Prednisolone Hemisuccinate Sodium Salt (MPS) Loading

MPS is a water soluble amphiphatic weak acid glucocorticosteroid prodrugused in the treatment of inflammation and autoimmune diseases.

MPS was loaded into LMVV using remote loading into pre-formed LMVVhaving a trans membrane calcium acetate (CA) gradient [see loading inClerc and Barenholz. (1995), BBA, 1240, 65-257, Avnir et al (2008)ARTHRITIS & RHEUMATISM, 58, 119-129)].

The loading was conducted under iso-osmotic conditions (0.5 ml LMVVincubated with 2 ml 25 mg/ml MPS plus sucrose to reach 280 mOsm). Theosmotic concentration inside and outside the liposomes were identical to280 mOsm/kg (similar to physiological saline).

LMVV Co-Loaded with BUP and MPS by Trans-Membrane CA Gradient

The procedure described above for BUP loading by CA gradient was usedusing a mixture of BUP and MPS under conditions that their mixture donot exceed iso-tonicity. Alternative active ingredients, such as otheramphiphatic weak acid steroid prodrugs, e.g. beta methaosonehemisuccinate may also be used.

Preparation of Ca⁺⁺ Cross-Linked Alginate Gel Beads Containing BupLoaded LMVV

The homogeneous blend solution (400 μl) contained fresh and cold (4° C.)2% (w/v) sodium alginate and BUP loaded LMVV (LMVV-BUP) 1:1 (V/V) wasdripped through a 25G*⅝″ (0.5 mm*16 mm) injection needle into 5 mlsolution of cold calcium chloride having an Osmolarity of 280 mOs whenLMVV prepared in 280 mOs were used or 550 mOs when LMVV prepared at 550mOs were used, as specified in each experiment

After 15 minutes of mechanical stirring at 4° C. smooth and sphericalbeads were formed. These beads were than washed with cold iso-osmoticNaCl solution (280 mOs when LMVV prepared in 280 mOs were used or 550mOs, when LMVV prepared at 550 mOs were used).

Each 5 beads (as manually counted) were incubated for a short time (<30minutes) with 50 μl iso-osmotic NaCl solution at 4° C.

When necessary to get de-crosslinking of the beads by Ca ions removal.small aliquot of 100 mM EDTA to be in excess to the Ca ions was added.[Meera George, T. Emilia Abraham, Journal of Controlled Release 114(2006) 1-14].

Preparation of Oxalate Cross-Linked Chitosan Gel Beads Containing BUPLoaded LMVV

LMVV loading was performed using transmembrane ammonium sulfategradient. LMVV of two lipid compositions were used: (i) HSPC100/CHOL(60:40 mole ratio) and (ii) HSPC100/C16SPM/CHOL (30:30:40 mole ratio).

Equal volumes of 2% (weight per water volume) chitosan solution and BUPloaded LMVV (which were not washed from the intersticial BUP) were mixedat 4° C. The mixtures were dripped into 120 mM (280 mosmomolar) oxalatefollowed by washing in cold saline. The beads formed were stored at 0.2;0.5; 1.0; or 2.0% bupivacaine solutions brought to 280 mOs with NaCl andstored in such storage media at 4° C. till their use. Then thesupernatant of beads was disposed.

Alternative Procedure for Storage of Cross Linked Hydrogel ContainingDrug Loaded LMVV:

The following alternative procedure was aimed to eliminate almostcompletely the issue of drug release during storage at 4° C.

To this end, LMVV were prepared and loaded with drug such as bupivacaine(without free drug removal) as described above. The LMVV wereconcentrated by one step centrifugation, the supernatant was removed andpacked LMVV were encapsulated in cross linked Ca alginate or chitosanoxalate hydrogel as described above.

The excess solution of CaCl₂ was replaced with the supernatant (storagemedia contained bupivacaine such as 0.2; 0.5; 1.0; or 2.0% bupivacainebrought to 280 mOs with NaCl) and stored in such storage media at 4° C.till its use.

Prior to injection for pain reduction or other indications (i.e.inflammation) the excess storage medium was either removed or notremoved as described in the specific experiment and the cross linkedhydrogel was either used as is or after being washed by 280 mOs NaCl asdescribed in the specific experiment.

Assays

Phospholipids (PL) determination: PL concentration was determined by themodified Bartlett method (Shmeeda et al., (2003), Method Enzymol. 367,272-292).

Bupivacaine determination: free (non liposomal) or total (liposomal plusfree) concentration of bupivacaine was measured by HPLC (Grant et alParm. Res., 2001 18, N3, 336-343; Anesthesiology, 2004, 101, 133-137).

Drug/lipid ratio determination for hydrogel beads: The washed beads wereinjected through a 25G×⅝″ (0.5 mm×16 mm) syringe needle to havehomogenous dispersion and were diluted ×10 with saline:

-   -   50 μl and 100 μl used for PL. determination.    -   100 μl centrifuged 10′ 2000 g at 4° C. and the supernatant        diluted ×10 with isopropanol (IPA) to determine free        bupivacaine.    -   100 or 200 μl heated in boiling water, than centrifuged 10′ at        2000 g. The supernatant was diluted ×10 with IPA to determine        total bupivacaine by HPLC.

All samples for bupivacaine determination were centrifuged for 2 min. at5000 g (using Eppendof centrifuge) after dilution in IPA. The upperphase was removed and analyzed by HPLC.

It is noted that the different batches of the same experiment may varyin terms of the volume of storage medium and this difference may affectthe absolute % of agent in the medium. However, as shown in the results,the general trend obtained from all batches is the same and thus theconclusion drawn from the various experiments is the same, namely, thatthe embedment of the liposomes in a hydrogel significantly reduces theagent leakage into the storage medium.

LMVV and MLV size distribution analysis: a particle size analyzer(Beckman Coulter LS 13 320) was used. This instrument combines twomethods of size distribution analyses: multi-wave light diffraction andpolarization intensity differential scattering (PIDS polarization of thelight). This combination allows the determination of broad sizedistributions in the range of 40 nm to 2 mm and therefore it is moresuitable for the size distribution analyses of large liposomes such asLMVV and large MLV.

Osmomolarity measurement: The osmomolarity of the solutions (storagemedia) was measured by ADVANCED INSTRUMENTS, Inc model 3320 osmometer(Norwood, Mass., USA).

Analgesic efficacy in male Swiss Webster mouse model: Testing foranalgesia was done by electrical stimulation of mice skin as describedelsewhere (G. J. Grant et al, Pharmaceutical Research, 18, no 3,336-343, 2001). Pain as electrical stimulation at the desired intensitywas inflicted to the skin of shaved mice abdomen. The current generator(model S48, Grass Instruments (W. Warwick, R.I. USA) was used. Mice(male Swiss-Webster, 26±3 gr (n=7 per group) were used. The mice wereshaved the hair overlying the abdomen and tested prior to injection todetermine the Individual vocalization threshold of each mice. Than themice were injected with (0.3-0.4 ml of one of the following: freebupivacaine, or LMVV-BUP or various LMVV entrapped in Ca-alginate orchitosan-oxalate cross linked hydrogels through 30G needle. Theanalgesia duration was than followed as specified in the experimentitself. All experiments were approved and ratified by the HUJI ethiccommittee.

Analysis of the in vivo mice analgesia: A numerical score to thespreadsheet was introduced for the evaluation of the analgesic effect ofvarious liposome preparations performance in vivo: For each time period(e.g. 4 hrs, 8 hrs etc) a numeric value to each mouse in the experimentwas given. If the anesthesia was complete the numeric value given was1.0; when analgesia was partial (incomplete) it received the numericvalue of 10.0 in the Table 1 and in other Tables a score of 0.5 and forno anesthesia the numeric score was 0. The mean for each subgroup wascalculated separately (i.e. 1% 300 μl, 2%150 μg).

Results Example 1 Characterization of LMVV-BUP

Bupivacaine to Lipids Ratio

BUP was remote loaded into LMVV by AS gradient using three different ofBUP to lipid v/v ratios:

-   -   (i) wet LMVV pellet to 5.7% BUP, using 1:4 vol/vol ratio.    -   (ii) wet LMVV pellet to 5.7% BUP, using 1:2 vol/vol ratio    -   (iii) wet LMVV pellet to 5.7% BUP using 1:1 vol/vol ratio.

The characteristics of the resulting LMVV are provided in Table 1:

TABLE 1 BUP loaded LMVV PL/CHOL ratio Loading method Mean size (μm)SPM/CHOL 6/4 CA gradient 8.33 ± 4.71 SPM/CHOL 6/4 AS gradient 5.7 ± 2.6HSPC/CHOL 6/4 passive 6.0 ± 3.2

FIGS. 1A-1B describe the release of Bupivacaine (BUP), during storage at4° C. Specifically, in FIG. 1A LMVV is composed of HSPC/CHOL (asdescribed in U.S. Pat. No. 6,162,462); HSPC/C16SPM/CHOL (3/3/4);HSPC100/CHOL (6/4) an HSPC100/C16SPM/CHOL (3/3/4) all ratios are moleper mole. Also shown, is the release of BUP at 37° C. (FIG. 1B) whereLMVV is composed of HSPC100/CHOL (6/4); HSPC100/C16SPM/CHOL (3/3/4), or,HSPC/C16SPM/CHOL (3/3/4), again, all ratios are mole ratio, All LMVVused have been loaded with BUP using an remote loading driven bytransmembrane ammonium sulphate (AS) gradient.

The data presented in FIG. 1A show that the release rates of BUP during60 days storage at 4° C. of the HSPC/CHOL LMVV (an identical formulationdescribed in U.S. Pat. No. 6,162,462) was the highest, followed by therelease rate from HSPC100/CHOL liposomes. The lowest release rate wasachieved when HSPC100/C16SPM/CHOL LMVV were used. FIG. 1B shows that in24 hours, the release at 37° C. of LMVV of the 3 compositions is almostidentical and reached the level of 65% to 70% of the LMVV BUP—stillwithout reaching a plateau. It was thus concluded that although a slightlower loading of BUP (lower BUP/PL ratio) was obtained with the LMVVcomposed of HSPC100/C16SPM/CHOL, their lowest release rate of BUP fromthis particular formulation at 4° C. without compromising it releaserate at 37° C. rendered this combination a preferred LMVV formulation.

The release rate from liposomes comprising HSPC100/C16SPM/CHOL 3/3/4(either SUV or LMVV as indicated) employing the different loadingtechniques, different active agents (BUP or MPS, the “Drug”) anddifferent aqueous media (washing and storing buffer) were examined. Theresults are presented in Table 2.

TABLE 2 Drug to phospholipid (PL) mole ratio and loading method effecton stability (defined as drug release at 4° C.) of LMVV using liposomes(LMVV or SUV composed of HSPC100/C16SPM/CHOL 3/3/4). Aqueous Drug/PL %free drug release at 4 c. Liposome type Loading technique medium moleratio 17 d 21 d 35 d 40 d 76 d 90 d 120 d 4.5 month 6 month LMVV-BUPPassive by 4.5% bup Saline 2% bup 1.5 17.8 LMVV-BUP Passive by 5.5% bupSaline 2% bup 1.7 20.9 36.3 LMVV-BUP Passive by 6% bup Saline 2% bup 1.723.5 36 LMVV-BUP Passive by 7% bup Saline 2% bup 1.9 25.8 40.6 LMVV-BUP250 mM CA grad PBS, 1% bup 0.8 11 19.9 44 LMVV-BUP 107 mm CA grad Saline0.6% bup 1.2 9 36.2 LMVV-BUP 107 mm CA grad Saline 0.7% bup 1.1 7.5 43LMVV-BUP 250 mm AS grad Saline 2% bup 1.6 8 11.1 21 LMVV-BUP 250 mm ASgrad 1.75% NaCl 1.4 2.5 9.9 22 LMVV-BUP 250 MM AS grad 1.75% NaCl 2 8 9LMVV-BUP 127 mm AS grad Saline 0.9% bup 2.3 3 9.6 13 LMVV-BUP 127 mm ASgrad Saline 0.7% bup 1.5 2.8 13.5 LMVV-BUP 127 mm AS grad Saline 1.5 3.320 LMVV-MPS 107 mm CA grad Saline 0.6 1.4 SUV-MPS 250 mm CA grad Saline0.3 LMVV-BUP 127 mm AS grad Saline 1.35 5 9 11.1 20 ml LMVV-BUP (20 127mm AS grad Saline 1.56 3.3 ml dialysis tube LMVV-BUP 10 127 mm AS gradSaline 1.17 ml diafiltration

FIGS. 2A and 2B demonstrate the release rate at 4° C. (FIG. 2A) and 37°C. (FIG. 2B) of BUP from LMVV having the same lipid compositions as usedin FIGS. 1A and 1B, wherein BUP was remotely loaded using Ca acetategradient. The SPM used was C16 SPM.

The ratio BUP/PL obtained by the CA gradient remote loading was lowerthan that obtained for the AS gradient remote loading. Stabilityassessed from the BUP release at 4° C. was lower than for the LMVV ofthe same lipid composition remote loaded by AS gradient (release ratewas higher) (Compare FIGS. 1A and 2A). At 37° C. the release rates aresimilar to those of the LMVV loaded with BUP by AS gradient, except thatrate of release is faster at the first 10 hours followed by an almostplateau (compare FIGS. 1B and 2B). It is apparent from FIG. 2A that theHSPC100 LMVV has best stability (i.e. lowest leakage at 4° C.) than HSPCbased LMVV, and that C16SPM effect on improving stability is muchgreater than the difference between the two HSPC preparations. C16SPMalso reduces leakage rate for both HSPC and HSPC100 compositions by asimilar extent.

FIGS. 3A and 3B demonstrate the release rate at 4° C. (FIG. 3A) and 37°C. (FIG. 3B) of BUP loaded LMVV of the same lipid compositions used inFIGS. 1A and 1B, wherein LMVV were passively loaded with BUP. The SPMused is C16SPM, and a comparison of HSPC/SPM/CHOL and HSPC100/SPM/CHOLwas also made at 4° C.

In general, release rates at 4° C. for passively loaded LMVV of the 3liposomal compositions used, were higher than for the remote loading viaCA gradient and much higher when compared with BUP AS remote loadedLMVV.

However the effect of LMVV lipid composition on release rates at 4° C.and 37° C. were similar (but larger in magnitude) to that observed forthe remote loading driven by AS or CA gradient, thus indicating that theion gradient driven remote loading increases LMVV loading stability at4° C.

LMVV Optimization

Various LMVV formulations with different mole ratio of HSPC100 to C16SPMwere prepared in order to determined the optimal mole ratio betweenthese two constituents. The different formulations are provided inTables 3A and 3B.

TABLE 3A Effect of HSPC100:C16SPM mole ratio in HSPC100/C16SPM/CHOL LMVVformed by active loading with AS gradient % SPM/ BUP HSPC100 BUP/PL %drug release at 4° C. at the specified times load- mole mole 2 3.5 ingratio ratio 8 d 22 d 30 d 38 d month month 4.5 0/1 2.2 2.5 8.2 18.9 4.51/0 1.8 4 9.5 15.5 4.5 1/1 1.68 8 5.7 1/1 1.96 7.5 8.7 5.7 5/4 2.03 5.27 5.7 2/1 1.5 5.8 7.8 5.7 7/2 1.6 5.3 7.5 5.7 0/1 1.8 4.3 5.7 1/1 1.552.6 5.7 2/1 1.44 2.4

TABLE 3B Effect of HSPC100 to C16SPM mole ratio in HSPC100/C16SPM/CHOLLMVV formed by active loading with CA gradient. % SPM/ BUP HSPC100BUP/PL % drug release at 4° C at the specified times load- mole mole 23.5 ing ratio ratio 8 d 22 d 30 d 38 d month month 4.5 0/1  1.7 2 19.241.2 4.5 1/0  1.45 7.4 8.8 20.8 4.5 1/1  1.77 15 4.5 0/1* 1.16 2 25.84.5 1/1* 1.5 3 12.6 34 4.5 1/3* 1.5 3.7 16 41 *HSPC and not HSPC100

Further, pre-formed LMVV were centrifuged for 5 min at 4° C. at 2000 gto give packed LMVV. For remote loading the packed LMVV were suspendedin various volumes of 5.7% BUP. The volume ratio of BUP to LMVV or PL isgiven in Table 4.

TABLE 4 Optimization of passive loading to the volume ratio of 5.7% BUPto packed LMVV (during loading). BUP/LMVV % free BUP volume ratio*BUP/PL mole ratio t = 0 4 1.17 0.4 2 1.23 0.6 1 1.13 2.8 Percent of BUPrelease from various LMVV formulations with or without C16SPM is alsopresented in FIGS. 7A to 7E. In these figures, the percent of free BUPstorage media at 4° C. or 37° C. is presented (1% is equivalent to 10mg/ml).

Percent of BUP release from various LMVV formulations with or withoutC16SPM is also presented in FIGS. 7A to 7E. In these figures, thepercent of free BUP storage media at 4° C. or 37° C. is presented (1% isequivalent to 10 mg/ml).

(B) Characterization of Alginate Gel Beads Encapsulated BupivacaineLoaded LMVV (ALG-LMVV-BUP)

Table 5 below provides characterization of the alginate beads embeddingLMVV-BUP-using LMVV composed of HSPC100/C16SPM/CHOL 3/3/4 (mole ratio).It is noted that compositions comprising MPS were prepared in a similarmanner using CA transmembrane gradient.

TABLE 5 Characterization of low viscosity alginate beads encapsulatingbupivacaine remote loaded LMVV (by ammonium sulfate transmembranegradient) LMVV-BUP beads EMPTY beads WEIGHT (mg/bead) 4.27 ± 0.6 3.3WATER mg/bead 3.085 ± 0.21 2.54 mg/mg bead 0.903 0.762 Ca⁺⁺ μg/bead3.906 ± 0.06 4.35 μg/mg beads 1.33  1.64 Phospholipids nmole/bead 51.34± 0.93 0 nmole/mg beads 14.88  Alginate μg/bead 22.7 ± 0.5 Bupivacainenmole/bead 68.06 ± 9.7  0 μg/bead 22.66 ± 3.3 

In addition, the kinetics of drug release (BUP or MPS) from the systemwere determined. For this the storage medium of Ca alginate hydrogelwere collected and the level of either BUP (as describes in Grant et al2004 ibid) or for MPS (as described in Avnir et al 2008 ibid) weredetermined using HPLC methods. The results are shown in Table 6.

TABLE 6 The kinetics of drugs (BUP or MPS) release from alginateencapsulated LMVV (HSPC100/C16SPM/CHOL 3/3/4) beads. LMVV loading % Freedrug release at 4° C.*** Sample process 16 d 22 d 39 d 60 d 74 d 100 dBUP-LMVV bead* Passive 4.5% 9.4 18.1 BUP-LMVV bead* Passive 5.5% 9.318.7 BUP-LMVV bead* Passive 6% 17.0 22.2 BUP-LMVV bead* Passive 7% 12.421 BUP-LMVV bead* 250 mM CA grad 10.0 15.8 BUP-LMVV bead* 250 mM AS grad11.8 15.6 BUP-LMVV bead* 127 mM AS grad 1.6 7.6 BUP-LMVV bead* 127 mM ASgrad 2.8 7.3 BUP-LMVV bead* 250 mM AS grad 7.2 11.5 BUP-LMVV bead* 250mM AS grad 2.7 13.1 BUP-LMVV bead* 127 mM AS grad 18 MPS-LMVV bead* 107mM CA grad 3.3 MPS-SUV bead* 107 mM CA grad 1.1 BUP-LMVV bead** 127 mMAS grad 1.5 8.5 *low viscosity alginate (LV ALG) **very low viscosityalginate (VLG ALG) ***only free drug release from the ALG-LMVV beads(the phospholipid determination in the liquid on the top of the beadswas 0, below detection limits).

Similar results are also presented in FIGS. 4A-4B showing the kineticsof BUP release from LV-ALG encapsulated LMVV (HSPC100/C16SPM/CHOL 3/3/4)beads at 4° C., 25° C. and 37° C. Specifically, FIGS. 4A and 4B showthat the release rate of BUP from the LMVV embedded in ALG beads at 37°C. reaches plateau after about 24 hours. The of BUP release rates ofALG-LMVV-BUP are similar to those of the LMVV loaded BUP (FIGS. 1A, 1B;2A, 2B and 3A, 3B), suggesting that at physiological temperature of 37°C. the encapsulation of the LMVV-BUP in the cross linked Ca alginatehydrogel did not affect BUP release rate namely, it would not reduce theefficacy of the formulation at use. Further, ALG-LMVV-BUP system basedon LMVV composed of HSPC100/C16SPM/CHOL 3/3/4 (mole ratio) exhibited lowrelease rates at 4° C. Thus, the LMVV-BUP was stable during 4° C.storage.

Further, MPS release from LV-ALG embedded LMVV (HSPC100/SPM/CHOL 3/3/4)or LV-ALG embedded SUV (HSPC/CHOL/PEG) at 37° C., into the aqueousstorage medium, was examined. Specifically, the following results wereobtained:

(I) ALG-LMVV-MPS beads: the release rate of free MPS (free form) fromALG after 1 week storage at 37° C. was 70%. This rate is slower fromthat achieved for ALG-LMVV-BUP beads (without being bound by theory,this probably related to the higher octanol to buffer distributioncoefficient or log D). These results are also comparable with the valuesobtained with the BUP formulation described elsewhere (FIG. 1, in Grantet al 2001, Pharma Res. 18 336-343), and with the values for MPS(described in Table 1 in Avnir et al 2008 ibid) which enables its fastertrans-membrane diffusion.

(II) ALG-SUV-MPS beads: This system showed a very different behaviorfrom the ALG systems encapsulated LMVV as it demonstrate the release ofintact SUV-MPS and not free MPS. This was measured by the release of MPSand phospholipid together. This is very different from the situationwhen LMVV-drug is used where only drug is released into the storagemedium and not any phospholipid (see above) During incubation onALG-SUV-MPS at 37° C. 8.7% of SUV-MPS were released to the storagemedium after 24 hours and 75% SUV-MPS was released after 1 week.

These results show that there is a size limitation for retaining theliposomes within the Ca alginate cross linked hydrogel. While SUV-MPSwere released from the hydrogel, LMVV were safely retained therein.

It should be noted that disposal of the aqueous storage medium in whichthe LV-ALG embedded LMVV or LV-ALG embedded SUV was stored and additionof fresh storage medium did not lead to substantial further release ofMPS to the storing medium, i.e. the level of free MPS in the freshmedium was almost zero.

FIG. 7, FIGS. 9A-9Q, FIGS. 10A-10E and FIG. 11A-11E describe the basicrelevant features of the ALG-LMVV-BUP system.

Specifically, FIG. 7 shows the kinetics of bupivacaine release at 37° C.from LMMV composed of HSPC100/C16SPM/CHOL (3/3/4 mole ratio) embeddedin: alginate hydrogel (ALG) cross-linked with Ca⁺⁺ ions (referred to asALG bupigel #3 and #4, as defined in Example 2 hereinbelow), ALG bupigelde-crosslinked with oxalic acid (ALG bupigel+OA, #5 as defined inExample 2 hereinbelow), chitosan (CHT) cross-linked with oxalate(referred to as CHT bupigel #6 or #7 as defined in Example 2hereinbelow) and CHT bupigel de-crosslinked with CaCl₂ (#8 CHTbupigel+CaCl₂ as defined in Example 2 hereinbelow).

describe and compare the change in level of free Bup (% of free Bup instorage media) during storage at 4° C. of LMVV (HSPC100/C16SPM/CHOLhaving 3/3/4 mole ratio) loaded with Bup using AS trans-membranegradient embedded in Ca-crosslinked alginate hydrogel (ALG-Beads) whenstored in various storage media. FIGS. 9A-9B shows the average effect ofstorage (6 months) in saline, 0.2%, 0.5%, or 2.0% Bup solutions (averageof different batches of ALG-Beads produced according to the methods ofthe invention). FIG. 9C shows the average effect of storage (3 months)in saline 0.5%, 2.0% Bup solutions. FIGS. 9D to 9F describe and comparethe change in level of free Bup (% free Bup in the different storagemedia) during storage at 4° C. of the different batches (herein denotedas A, B, C, D, E and an average plot) of LMVV (HSPC100/C16SPM/CHOLhaving 3/3/4 mole ratio) loaded with Bup using AS trans-membranegradient embedded in Ca-crosslinked alginate hydrogel when stored invarious storage media, during 3 months: saline (FIG. 9D), 0.5% BUP (FIG.9E) and 2.0% BUP (FIG. 9F). FIGS. 9G-9K describe and compare the changein level of free Bup (% of free Bup in the different storage media)during storage at 4° C. of the different batches (herein denoted as A,B, C, D, E and an average plot) LMVV (HSPC100/C16SPM/CHOL having 3/3/4mole ratio) loaded with Bup using AS trans-membrane gradient embedded inCa-crosslinked alginate hydrogel when stored in various storage media,during 3 months in: saline (FIG. 9G), in 0.2% BUP (FIG. 9H), in 0.5% BUP(FIG. 9I) or in 2.0% BUP (FIG. 9J). FIG. 9K describes the average effectof storage (3 months) in saline, 0.2%, 0.5%, or 2.0% Bup solutions(average of different batches of ALG-Beads described in FIG. 9G to 9J).FIG. 9L shows the average effect of storage (2 months) in saline 0.5%,2.0% Bup solutions. FIGS. 9M-9Q describe and compare the change in levelof free bupivacaine (% of free Bup in storage medium) of LMVV(HSPC100/C16SPM/CHOL having 3/3/4 mole ratio) loaded with Bup via the AStrans-membrane not embedded in hydrogels when stored in various storagemedia at 4° C. (FIGS. 9M, 9N and 9O in saline, 0.5%, or 2.0% Bupsolutions and FIG. 9P in saline, 0.2%, 0.5%, or 2.0% Bup solutions).FIG. 9Q describes a separate experiment of 2 months follow-up uponstorage in saline without hydrogel. All storage media in FIGS. 9A-9Qwere brought to 285 mOsmole by addition of NaCl solution to retainiso-tonicity

FIG. 10A-10D describe the change in level of free Bup (% of free Bup instorage media) in different storage media of LMVV(HSPC100/C16SPM/CHOL3/3/4 mole ratio) loaded with Bup via trans-membraneAS gradient and embedded in Ca cross-linked alginate. Three storagemedia were used (saline, 0.5% Bup, and 2.0% Bup, all storage media werebrought to iso-tonicity of 285 mOsmole with NaCl solution). These werestored for 40 days at 4° C. and than used in the experiments describedin FIG. 10A-10D). FIGS. 10A and 10B describe release of Bup after theremoval of storage media by washing with saline followed by 30 hours ofincubation at 37° C. throughout this time amount of drug in the hydrogelmedia (saline) was measured (FIG. 10A) and also described as % Bupreleased (FIG. 10B). FIG. 10C describes the change in Bup level in thestorage media of the preparations incubated in their original storagemedia (0.5 and 2.0% Bup) at 37° C. for 25 hours. FIG. 10D is anextension of data from FIG. 10C to 15 days of incubation, FIG. 10E showsthe change in Bup concentration in 0.5% Bup, and 2.0% Bup storage mediaof the above described Bup loaded LMVV after incubation at a temperatureof 25° C. for 20 days.

FIGS. 11A-11E describe change in level of free Bup (% of free Bup instorage media) over a storage period of 3 months at 4° C., in differentliquid storage media of ALG-LMVV-BUP (HSPC100/C16SPM/CHOL 3/3/4 moleratio), remote loaded by trans-membrane AS gradient. The storage mediaused were: Saline (FIG. 11A), 0.2% BUP (FIG. 11B), 0.5% BUP (FIG. 11C),and 2.0% BUP (FIG. 11D). FIG. 11E summarizes the changes in Bupconcentration over a storage period of 3 months at 4° C., in differentliquid storage media of CHT-LMVV-BUP (HSPC100/C16SPM/CHOL 3/3/4 moleratio), remote loaded by trans-membrane AS gradient. The storage mediaused were: Saline, 0.2% BUP, 0.5% BUP, and 2.0% BUP.

In Vivo Experiments

Bupivacaine Loaded LMVV Preparations

FIGS. 5A-5C and 6A-6F summarize results obtained with 8 LMVV-BUPformulations. These were prepared (as specified below) under sterileconditions and were tested for sterility in the Clinical MicrobiologyDepartment, Hadassah Hospital, Jerusalem, Israel. These liposomes werealso characterized for their size distribution, drug to PL mole ratioand rate of BUP release at 4° C. and 37° C. The liposomes were shippedfrom Jerusalem Israel to Dr G. J. Grant, Department of Anesthesiology,NYU, School of Medicine, NYC, USA at controlled temperature of 2° C.-8°C. Using sensors attached to the shipped samples it was found that thetemperature was kept at the desired rang suring shipment. HPLC analysisbefore shipment and after arrival to destination indicated that noleakage during shipment took place. This is described as “In vivoExperiment 1” or NYU experiment.

TABLE 7 Characterization of liposomes composition and properties offormulations used in in vivo experiment 1 Ratio y = 141.555xBupivicaine/ pellet total % of Bupivicaine Pi Pi mM date of sampleliposomes volume volume free (total) μmol/ml = Bupivicaine/ samplepreparation number gradient sort type ml ml bupiv. mM mmol/l = mM mM PiH100/SPM_(C16)/CHOL 15 Jul. 2007 1 AS (in saline) MLV 3.5 15 5.08 17.1128.12 0.61 3/3/4 1 ml lipos (instead 0.5 ml) + 2 ml 4.5% bup. 09 Jul.2007 2 CaAc MLV 4 15 3.09 17.86 19.53 0.91 (in PBS) 10 Jul. 2007 3 ASLMVV 5 15 2.81 27.36 13.71 2.00 (in saline) 11 Jul. 2007 & 4 CaAc LMVV15 30 3.56 17.28 21.06 0.82 15 Jul. 2007 (in PBS) H100/CHOL 16 Jul. 20075 AS LMVV 7 15 3.27 32.23 15.91 2.03 6/4 (in saline) HSPC/CHOL 16 Jul.2007 6 AS LMVV 6 15 6.81 33.32 14.89 2.24 6/4 (in saline) H100/CHOL 17Jul. 2007 7 CaAc LMVV 7 15 6.11 14.67 19.82 0.74 6/4 (in PBS)H100/SPM_(C16)/CHOL 18 Jul. 2007 8 6% passive LMVV 4 15 1.20 23.00 20.131.14 3/3/4

All liposomal formulations were analyzed for free non liposomal BUP andtotal BUP before the in vivo experiment followed by their concentrationto reach the level of 2% (w/w) BUP (for liposome formulations #1, 2, 3,5, 6, 8) and to 1% (w/w BUP for liposome formulations #4 & 7). BUP wasloaded into the liposomes either by active loading (CA or AS gradient)or by passive loading as described in Table 8.

TABLE 8 Liposome characterization prior to Experiment 1 performanceLiposome % free # Lipids Loading technique type BUP 1 H100/SPM/CHOL ASgradient MLV 3.88 2 H100/SPM/CHOL CA gradient MLV 3.95 3 H100/SPM/CHOLAS gradient LMVV 3.69 4 H100/SPM/CHOL CA gradient LMVV 4.52 5 H100/CHOLAS gradient LMVV 3.68 6 HSPC/CHOL AS gradient LMVV 7.80 7 H100/CHOL CAgradient LMVV 7.66 8 H100/SPM/CHOL 6% BUP passive loading LMVV 1.90Analgesic Efficacy in Mouse Model:

Testing for analgesia was done by electrical stimulation of the skindirectly overlying the abdomen at the site of injection using a currentgenerator (model S48, Grass Instruments) as described in Grant et al2001. (G. J. Grant et al, pharmaceutical research, vol 18, no 3,336-343, 2001), and in Methods above.

Mice (male Swiss-Webster 26±3 gr) were tested prior to injection todetermine the vocalization threshold than were injected with liposomalBUP or free BUP than followed by determination of analgesia duration(The duration of the main in vivo screening study was 2 days and startedafter a preliminary study using two different injection volumes offormulation #4 (referred to as the PILOT in Table 9A) was performed.

In order to evaluate the effect of altering the volume and BUPconcentration of the injection, in each of the 7 groups (all groupsexcept group 4) three mice received 150 μl of the 2% formulation and 3mice received 300 μl 1.0% (achieved by a dilution of the 2%formulation).

It has been previously determined (Grant et al. 2004, ibid., and U.S.Pat. No. 6,162,462) that free (standard, non liposomal) BUP provide ananalgesic effect for approximately 75 minutes post injection.

The analgesic efficacy of the various formulations 1 to 8, at differentBUP concentration, different injection volume etc. is presented inTables 9A to 9C. In these Tables, an numeric score of “1” denotes fullanalgesia, a numeric score of “0” was given when there was no analgesiceffect, and a numeric value of “10” when there was partial analgesia.

In Table 9A results of mice injected with LMVV formulation #4, two micewith 300 μl and two mice with 150 μl are presented as “PILOT 1-4”Testing was done at 4, 17, and 21 hours following injection.

In all other 7 mice groups the mice were injected with either 300 μl ofliposomes containing 1.0% BUP or 150 μl of liposomes containing 2.0%BUP. So total BUP injected per mouse was the same (3 mg/mouse).

The results of the pilot study are also described in Table 9A.

TABLE 9A Duration of analgesia at different BUP concentrations(administered as liposomal-BUP) and different injected volumes Aug. 9,2007 1 indicates mice under analgesia, 0 indicates mice lacks analgesia;10 indicates mice is under partial analgesia Note: On Aug. 8, 2007, weinjected four animals with LMW formulation #4 (2 animals with 300 ul and2 mice with 150 ul); testing was done at 4, 17, and 21 hours. These arelabeled “PILOT” in the spreadsheet below animal # lipo # bup conc volume(ul) mg Bup 4 hr 8 hr 12 hr 15 hr 18 hr 21 hr 1 1 2% 150 3 1 1 10 0 0 02 1 2% 150 3 1 1 1 1 0 0 3 1 2% 150 3 1 1 1 1 0 0 4 1 1% 300 3 1 1 1 0 00 5 1 1% 300 3 1 1 1 0 0 0 6 1 1% 300 3 1 1 1 0 0 0 7 2 2% 150 3 1 1 1 10 0 8 2 2% 150 3 1 1 0 0 0 0 9 2 2% 150 3 1 1 1 0 0 0 10 2 1% 300 3 1 11 1 0 0 11 2 1% 300 3 1 1 0 0 0 0 12 2 1% 300 3 1 1 0 0 0 0 13 3 2% 1503 1 1 1 1 0 0 14 3 2% 150 3 1 1 1 0 0 0 15 3 2% 150 3 1 1 1 1 0 0 16 31% 300 3 1 1 1 0 0 0 17 3 1% 300 3 1 1 1 1 1 0 18 3 1% 300 3 1 1 1 1 0 019 4 1% 300 3 1 1 1 1 1 0 20 4 1% 300 3 animal eliminated from study 214 1% 300 3 1 1 1 10 10 0 22 4 1% 300 3 1 1 1 1 10 10 23 4 1% 300 3 1 1 11 10 0 24 4 1% 300 3 1 1 1 1 0 0 17 hr PILOT 1 4 1% 300 3 1 1 0 PILOT 24 1% 300 3 1 1 0 PILOT 3 4 1% 150 1.5 1 0 PILOT 4 4 1% 150 1.5 1 0 25 52% 150 3 1 1 1 0 0 0 26 5 2% 150 3 1 1 1 1 1 0 27 5 2% 150 3 1 1 1 0 0 028 5 1% 300 3 1 1 1 1 0 0 29 5 1% 300 3 1 1 1 0 0 0 30 5 1% 300 3 1 1 100 0 0 31 6 2% 150 3 1 1 0 1 0 0 32 6 2% 150 3 1 1 1 1 0 0 33 6 2% 150 31 0 0 0 0 0 34 6 1% 300 3 1 1 0 0 0 0 35 6 1% 300 3 1 1 1 10 10 0 36 61% 300 3 1 1 1 0 0 0 37 7 1% 300 3 1 1 1 10 10 0 38 7 1% 300 3 1 1 1 1 00 39 7 1% 300 3 1 0 0 0 0 0 40 7 1% 300 3 1 1 0 0 0 0 41 7 1% 300 3 1 11 1 0 0 42 7 1% 300 3 1 1 1 1 0 0 43 8 2% 150 3 1 1 1 1 0 0 44 8 2% 1503 1 1 1 0 0 0 45 8 2% 150 3 1 1 1 0 0 0 46 8 1% 300 3 1 1 1 1 0 0 47 81% 300 3 1 1 1 1 0 0 48 8 1% 300 3 1 1 1 0 0 0

TABLE 9B Duration of analgesia at different free BUP concentrations andat different injected volumes Aug. 13, 2007 Standard Bupivacaine(Control) 1 = analgesia; 0 = no analgesia; 10 = partial analgesia Mouse#Bup Conc Volume (ul) mg Bup 15 min 30 min 45 min 60 min 75 min 90 min105 min 120 min 135 min 1 0.25% 150 0.375 1 1 1 0 0 0 2 0.25% 150 0.3751 1 1 0 0 0 3 0.25% 150 0.375 1 1 1 0 0 0 4 0.25% 150 0.375 1 1 1 1 0 05 0.25% 150 0.375 1 1 1 1 10 0 6 0.25% 150 0.375 1 1 1 0 0 0 7 0.25% 1500.375 1 1 1 10 0 0 8 0.25% 150 0.375 1 1 1 0 0 0 1 0.25% 300 0.75 1 1 11 0 0 0 0 0 2 0.25% 300 0.75 1 1 1 1 1 1 0 0 0 3 0.25% 300 0.75 1 1 1 11 10 10 0 0 4 0.25% 300 0.75 1 1 1 1 1 10 0 0 0 5 0.25% 300 0.75 1 1 1 11 1 1 10 0 6 0.25% 300 0.75 1 1 1 1 1 1 0 0 0 7 0.25% 300 0.75 1 1 1 1 11 0 0 0 8 0.25% 300 0.75 1 1 1 1 1 1 1 0 0 1 0.50% 150 0.75 1 1 1 1 1 00 2 0.50% 150 0.75 1 1 1 1 1 0 0 3 0.50% 150 0.75 1 1 1 1 1 10 0 4 0.50%150 0.75 1 1 1 1 0 0 0 5 0.50% 150 0.75 1 1 1 1 1 1 0 6 0.50% 150 0.75 11 1 1 1 10 0 7 0.50% 150 0.75 1 1 1 1 1 1 0 8 0.50% 150 0.75 1 1 1 10 100 0 Liposomal (LMVV) Bupivacaine Pilot Study Mouse # LipoForm# Conc.Volume mg Bup 15 hr 18 hr 21 hr 1 3 2% 300 6 1 1 10 2 3 2% 300 6 1 1 0 14 1% 450 4.5 1 1 0 2 4 1% 450 4.5 1 1 0 1 5 2% 300 6 1 1 0 2 5 2% 300 60 1 0

TABLE 9C Analgesic effect at different free BUP concentrations anddifferent injected volumes Aug. 13, 2007 Standard Bupivacaine (Control)1 = analgesia; 0 = no analgesia; 10 = partial analgesia Mouse # Bup ConcVolume (ul) mg Bup 15 min 30 min 45 min 60 min 75 min 90 min 105 min 120min 135 min 1 0.25% 150 0.375 1 1 1 0 0 0 2 0.25% 150 0.375 1 1 1 0 0 03 0.25% 150 0.375 1 1 1 0 0 0 4 0.25% 150 0.375 1 1 1 1 0 0 5 0.25% 1500.375 1 1 1 1 10 0 6 0.25% 150 0.375 1 1 1 0 0 0 7 0.25% 150 0.375 1 1 110 0 0 8 0.25% 150 0.375 1 1 1 0 0 0 1 0.25% 300 0.75 1 1 1 1 0 0 0 0 02 0.25% 300 0.75 1 1 1 1 1 1 0 0 0 3 0.25% 300 0.75 1 1 1 1 1 10 10 0 04 0.25% 300 0.75 1 1 1 1 1 10 0 0 0 5 0.25% 300 0.75 1 1 1 1 1 1 1 10 06 0.25% 300 0.75 1 1 1 1 1 1 0 0 0 7 0.25% 300 0.75 1 1 1 1 1 1 0 0 0 80.25% 300 0.75 1 1 1 1 1 1 1 0 0 1 0.50% 150 0.75 1 1 1 1 1 0 0 2 0.50%150 0.75 1 1 1 1 1 0 0 3 0.50% 150 0.75 1 1 1 1 1 10 0 4 0.50% 150 0.751 1 1 1 0 0 0 5 0.50% 150 0.75 1 1 1 1 1 1 0 6 0.50% 150 0.75 1 1 1 1 110 0 7 0.50% 150 0.75 1 1 1 1 1 1 0 8 0.50% 150 0.75 1 1 1 10 10 0 0Liposomal (LMVV) Bupivacaine Pilot Study Mouse # LipoForm# Conc. Volumemg Bup 15 hr 18 hr 21 hr 1 3 2% 300 6 1 1 10 2 3 2% 300 6 1 1 0 1 4 1%450 4.5 1 1 0 2 4 1% 450 4.5 1 1 0 1 5 2% 300 6 1 1 0 2 5 2% 300 6 0 1 0

The results of Table 9A-9C and FIGS. 5A-5C and 6A-6F show clearly thelarge superiority of all liposomal BUP (more than 8 hours for allliposomal 1-8 groups, in some of groups (#1, 2, 3, 4, 5, 7, 8 even 12hours of) over free BUP in which there is a complete disappearance ofanalgesia in less then 2 hours post administration. Comparing theperformance of the different liposomal-BUP formulations (described inTable 8) formulation 4, where BUP was actively loaded into LMVV viatransmembrane CA gradient and the stored at iso-osmotic aqueous salineprovided the longest analgesia effect. However the differences from theother formulations (#1, 2, 3, 5, 7, 8) was not significant especiallywhen compared to the large increase in analgesia duration when comparedto free BUP. These efficacy data are in agreement with those previouslydescribed for the unstable HSPC/CHOL LMVV formulations used by Grant etal. 2004, ibid., and U.S. Pat. No. 6,162,462].

In a separate experiment the effect of repeated injection of bupivacaineloaded LMVV in mice was evaluated. For this 3 mice were injected with150 μl of liposomes containing 2.0% BUP and level of analgesia wasdetermined as described in Table 9A, the results were similar to thosein Table 9A. After 15 hours a second (repeated) injection was given tothe same mice and the duration of analgesia was followed for another 24hours. The results showed that the analgesia obtained after the second(repeated) injection was identical to the one achieved at the firstinjection without any observed side effect. The conclusion was thatanalgesia can be prolonged by repeated injections and the time period ofanalgesia after the second injection was at least of the same durationas that obtained after the first injection, namely there was nointerference of the hydrogel.

Example 2: Efficacy of Hydrogels (Ca-Alginate and Chitosan-Oxalate)Encapsulating BUP Loaded LMVV

The method used in Example 2 are the same as described above.

Results

Formulation Preparations and Characterization:

The following 9 different formulations were prepared: All LMVV have thelipid composition of HSPC100:C16SPM:CHOL 3:3:4 mole/mole and were remoteloaded with BUP via AS gradient.

-   -   1—Bupivacaine loaded LMVV. Stored in saline at 4° C. for 16        days.    -   2—Alginate (VLG ALG) beads containing LMVV-BUP without free        bupivacaine removal and stored at 4° C. for 4 months in 5.7%        (iso-osmotic) bupivacaine (to prevent net drug leakage during        storage). Before injection to mice the beads washed 10 times        with 10 ml saline.    -   3—ALG beads containing LMVV-BUP (free drug was removed) and        stored at 4° C. in saline for 16 days.    -   4—ALG beads containing LMVV-BUP (after removal of free drug)        were stored at 4° C. in 5.7% bupivacaine for 16 days (to prevent        reduction of drug to lipid ratio during storage). Before        injection to mice these beads washed 10×10 ml of saline.    -   5—ALG beads containing LMVV-BUP stored at 4° C. in saline for 16        days. The beads were de-cross linked by 100 ul (each vial) 15        mg/ml oxalic acid (OA) before mice injection.    -   6—Chitosan (CHT) beads containing LMVV-BUP prepared without free        bupivacaine removal and stored at 4° C. for 4 months in 5.7%        bupivacaine (to prevent net drug leakage while storage). Before        injection to mice the beads washed with OA and 10 times with 10        ml of saline each.    -   7—CHT beads contained LMVV-BUP (free drug was removed) and        stored at 4° C. in saline for 16 days.    -   8—CHT beads contained LMVV-BUP and stored at 4° C. in saline for        16 days. The beads were de-cross linked by 100 ul 15.4 mg/ml        CaCl₂ before mice injection.    -   9—Free bupivacaine 0.25%.

TABLE 10a samples characterization (analyzed before starting theexperiment). Free bup concentration(%) after injection Total BUP Samplethrough G31 BUP PL. BUP/PL. concentration number syringe needle mM (mM)(mM) (%) 1 0.067 47.68 21.7 2.2 1.5  diluted to 1% 2 0.056 17.0 14.11.21 0.54 3 0.166 29.57 15.3 1.93 0.93 4 0.132 32.39 18.0 1.8 1.02 50.049 23.21 13.7 1.69 0.73 6 0.057 18.48 12.35 1.5 0.58 7 0.06 28.2112.56 2.25 0.89 8 0.087 28.21 0.89 9 0.25 0.25

TABLE 10b samples characterization (analyzed after the experiment wasfinished). Free bup concentration(%) after injection Total BUP Samplethrough G31 BUP PL. BUP/PL. concentration number syringe needle mM (mM)(mM) (%) 1 0.096 26.8 14.6 1.8 0.8 2 0.744 48.8 16.8 2.9 1.5 3 0.14630.8 16.3 1.88 0.95 5 0.06 25.9 14.7 1.76 0.8 6 0.312 41.8 17.6 3.2 1.37 0.125 50.2 23.3 2.1 1.5 8 0.12 31.5 15.2 2.1 0.97 9 0.25 0.25 Note:sample #2 and #6 had high free bupivacaine concentration.

TABLE 11 Bupivacaine release from beads' formulations at 37° C. time 0 2hours 4 hours 6 hours 15 hours 23 hours 24 hours % bup % bup % bup % bup% bup % bup % bup sample # release release release release releaserelease release 3 17.8 19.5 51.2 65.1 73.0 89.2 4 12.9 16.1 9.9 90.4 55.9 23.5 41.8 50.6 72.1 99.3 6 36.1 39.2 38.5 48.5 69.23 7 6.9 18.5 20.535.7 77.2 64.8 91.9 8 9.7 23.1 10.9 57.7 69.1 101.2 Conclusion: alldifferent HYDROGEL- LMVV-BUP formulations released the drug at 37° C. ina similar rate achieving total release after 24 hours.

TABLE 12A analgesia duration in mice mouse group 1 4 7 10 12 14 16 18 2022 24 26 27 28 29 30 32 34 49 # # hr h hr hr hr hr hr hr hr hr hr hr hrhr hr hr hr hr hr 1 1 1 1 1 1 1 1 1 0 0 2 1 1 1 1 1 1 1 0 0 3 1 1 1 1 11 1 0 0 4 1 1 1 1 1 1 1 0 0 5 1 1 1 1 1 1 1 1 0.5 0 6 1 1 1 1 1 0 0 0 71 1 1 1 1 1 1 1 0 0 1  2** 1 1 1 1 1 1 1 1 1 1 1 1 1 0 2  2** 1 1 1 1 11 1 1 1 1 1 1 1 0 3  2** 1 1 1 1 1 1 1 1 1 0 0 4  2** 1 1 1 1 1 1 1 1 11 1 1 0.5 0 5  2** 1 1 1 1 1 1 1 0 0 6  2** 1 0.5 1 1 1 1 1 0 0 7  2**dead 1 3 1 1 1 1 1 1 1 1 0 0 2 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 3 3 11 1 1 1 1 1 1 0 0 4 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 5 3 1 1 1 1 1 11 1 0 0 6 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 7 3 1 1 1 1 1 1 1 1 1 1 11 0 1  4** dead 2  4** dead 3  4** 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 4 4** 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 5  4** 1 1 1 1 1 1 0 0 6  4** 1 1 1 11 1 1 1 1 1 1 0.5 0 7  4** 1 1 1 1 1 1 1 0.5 0.5 1 1 1 0 1 5 1 1 1 1 1 11 1 0 0 2 5 1 1 1 1 1 1 1 1 1 0.5 1 1 0 0 3 5 1 1 1 1 1 1 1 0 0 4 5 1 11 1 1 1 0 0 5 5 1 1 1 1 1 1 1 0 0 6 5 1 1 1 1 1 1 1 1 0 0.5 1 0 0 7 5 11 1 1 1 0.5 0 0  1*  6** 1 1 1 1 1 1 1 1 1 1 1 1  2*  6** 1 1 1 1 1 1 11 1 1 1 1 0 0  3*  6** 1 1 1 1 1 1 1 1 1 1 1 1 4  6** 1 1 1 1 1 1 1 1 11 1 1  5*  6** 1 1 1 1 1 1 1 0 0.5 0  6*  6** 1 1 1 1 1 1 1 1 1 1 1 1 07  6** 1 0.5 1 7 1 1 1 1 1 1 1 1 0.5 0.5 1 1 1 1 1 0 2 7 1 1 1 1 1 1 1 11 1 1 1 1 1 1 0 3 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 4 7 1 1 1 1 1 1 1 11 0.5 1 1 1 1 1 1 5 7 1 1 1 1 1 1 1 1 1 1 1 1 6 7 1 1 1 1 1 1 1 1 1 0.51 1 1 1 1 1 7 7 1 1 1 1 1 1 1 1 1 1 1 1  1* 8 1 1 1 1 1 1 1 1 0.5 0 1 11 0  2* 8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0  3* 8 1 1 1 1 1 1 1 1 1 1 1 11 1 1 0 4 8 1 1 1 1 1 1 1 1 1 0.5 1 1 1 0 0 0  5* 8 1 1 1 1 1 1 1 0 0 11 1 0 6 8 1 1 1 1 1 1 1 1 1 1 1 1 0 0 7 8 1 1 1 1 1 1 1 1 1 0 1 0 0 1 =analgesia; 0 = no analgesia; 10 = partial analgesia; *irritation atinjection site; **toxic from the free bup

TABLE 12B (group 9 only (contiuation of Table 12A) standard freebupivacaine (group 9) mouse # 0.25% bup 5′ 30′ 60′ 75′ 90′ 105′ 120′135′ 150′ 1 300 ul 1 1 1 1 1 0 0 2 300 ul 1 1 1 0 0 3 300 ul 1 1 1 0 0 4300 ul 1 1 1 1 1 0 0 5 300 ul 1 1 1 1 1 1 0.5 0 0

TABLE 13 Toxic and side effects in mice storing formulation solution*effects descriptions 1-LMVV saline no mortality, no abnormal clinicalsigns, no obvious side effects. 2-ALG-bupigel 5.7% bup induced severesystemic toxicity with appearance of convulsions within 2 min after Inj.In 6 mice the damage was reversible after 1-2 hours. 1 mice died.3-ALG-bupigel saline no mortality, no abnormal clinical signs, noobvious side effects. 4-ALG-bupigel 5.7% bup induced severe systemictoxicity with appearance of convulsions within 2 min after Inj. In 5mice the damage was reversible after 1-2 hours. 2 mice died.5-ALG-bupigel saline no mortality, no abnormal clinical signs, noobvious side & oxalic acid effects. 6-CHT-bupigel 5.7% bup inducedsevere systemic toxicity with appearance of convulsions within 2 minafter Inj. In 7 mice the damage was reversible after 1-2 hours, in 5mice: skin irritation at inj. Site was found. 7-CHT-bupigel saline nomortality, no abnormal clinical signs, no obvious side effects.8-CHT-bupigel saline no mortality, no abnormal clinical signs, in 4mice: skin & CaCl2 irritation at inj. site. 2- ALG-bupigel 0.5% bup nomortality, no abnormal clinical signs, no obvious side effects (group of5 mice) *all beads formulations were washed from the storing solutionsbefore injection to the mice.

Control Groups:

Three control groups (5 mice per group) were studied:

The control groups aimed to check in the various formulations effectswhich are unrelated to bupivacaine but related to the formulation itselfor to the act of injection.

The three control groups included:

-   -   1—ALG beads made of mixture 250 ul 2% ALG solution in water and        250 ul saline than cross linking by CaCl2.    -   2—ALG solution: mixture of 250 ul 2% ALG and 250 ul saline.    -   3—CHT solution: mixture of 250 ul 2% CHT and 250 ul saline. (CHT        beads were very hard to pass through the needles 31G and 25G).

TABLE 14 analgesia duration in mice, controls (300 ul injection permice) blank, plymers (no bup) mouse # SAMPLE 5 min 30 min 1 hr 3 hr 5 hr9 hr 12 hr 14 hr 17 hr 19 hr 20 hr 24 hr 28 hr 38 hr 1 ALG BEADS 1 0 0 00 0 2 ALG BEADS 1 1 0 0 0 0 3 ALG BEADS 0 0 0 0 0 0 4 ALG BEADS 1 1 0.50.5 0.5 1 1 1 1 1 1 1 0 5 ALG BEADS 0 1 1 1 1 1 1 1 1 1 1 1 0 1 ALGBEADS 0 2 ALG BEADS 0 3 ALG BEADS 0 4 ALG BEADS 1 5 ALG BEADS 0 1 1% ALGsol. 0 2 1% ALG sol. 0 3 1% ALG sol. 0 1 1% CHT sol. 0 0 0 0 0 0 2 1%CHT sol. 0 0 0 0 0 0  3* 1% CHT sol. 1 1 1 1 1 1 1 1 1 1 1 1 0  4* 1%CHT sol. 1 1 1 1 1 1 1 1 1 1 1 0.5 0 5 1% CHT sol. 1 0 0.5 0 0 0

Mice 4 and 5 had analgesia from the ALG beads for 24 hours. Likewise,mice 3 and 4 which received chitosan sol. We have seen this phenomenonin the past, and currently we have no explanation for it.

Conclusions

All formulations (1 to 8) showed a prolonged analgesia compared with thefree bupivacaine.

All different Hydrogel LMVV-BUP formulations) release the drug at 37° C.in a similar rate achieving total release after 24 hours.

All HYDROGEL-LMVV-BUP in hydrogels showed prolonged analgesia overLMVV-BUP.

Two of the CHT-LMVV-BUP (formulation 7;8) showed the highest analgesiaprolongation followed closely by one of the ALG-LMVV-BUP (formulation 4)which was better than two other ALG-LMVV BUP formulations (3 & 5)followed by LMVV-BUP (formulation 1 which gave identical data to the asimilar LMVV-BUP used in in vivo experiment 1 (NYU) referred to in thefig legend as LMVV-BUP (NYU), free bupivacaine (formulation 9) showedthe shortest analgesia duration.

The beads of formulations 2, 4, 6 were stored at a high concentration offree bupivacaine (5.7%) in order to prevent change of drug to lipidratio during storage. Before injection the beads were washed with coldun-buffered saline (pH=5.5). However as 5.7% bupivacaine at pH 5.5,exceeds the solubility limit which at 25° C. is below 2.9% (the masssolubility of bupivacaine at 25° C. in pH 5 is 2.9% and in pH 6 is0.55%) bupivacaine may precipitate/crystallize. The solution of 5.7%bupivacaine in water was prepared by heating the solution close to waterboiling followed by cooling to 4° C. Under such conditions noprecipitation of bupivacaine occurs for few hours. In the hydrogel beadsformulations 2, 4 and 6 free 5.7% bupivacaine crystallize/precipitate onthe hydrogel fibers during storage at 4° C. and can not be washed by thecold saline. Therefore toxic effects in these formulations: severesystemic toxicity with appearance of convulsions within 2 min afterinjection. For most of the mice the damage was reversible after 1-2hours (the time that the free bup was remained).

Side effects may be reduced by one or more of the following:

-   -   Pre-wash of the beads with solution at lower pH (such as pH 2.0)        in which solubility of bupivacaine 25° C. is 7.5%.    -   Storing the LMVV-BUP at lower concentration of bupivacaine        (0.5%-3%) that should not precipitate or crystallized and        therefore can be washed by saline.    -   use of ultra pure grade chitosan and possibly chitosan of lower        viscosity to avoid or reduce irritation at the injection site in        some of CHT formulations.

Without being bound by theory, it is believed that the use of LMVVencapsulated within hydrogel beads either the negatively chargedpolysaccharide alginate or the positively charged Chitosan, reduces thelevel of free drug (non liposomal) in the administered product usingfast and easy washing of the beads. Intact LMVV are not released fromthe beads, while the free bupivacaine and even small liposomes arereleased. It is noted that hydrogen encapsulation did not affect drugrelease rate from LMVV. Chitosan, being positively charged, seems to actlike a bioadhesive and probably binds to negatively charged surfaceslike those of most cells, glycoproteins, cartilage etc, which may be anadvantage as suggested by this preliminary study. It is known to bebiocompatible.

Example 3: Scale Up of ALGINATE-Bup Loaded LMVV Beads

The following example is aimed providing:

1—Scale-up of LMVV preparation to 10 ml and 100 ml, using lipidcomposition of HSPC100/C16SPM/CHOLESTEROL 3/3/4 and ammonium sulfate forloading bupivacaine under iso-osmotic conditions.

2—Improve shelf life of LMVV-BUP by encapsulation of the bupivacaineloaded LMVV in cross-linked polymer hydrogel beads using low viscosityalginate (negative charge).

3—Evaluation of the efficacy of ALG-LMVV-BUP BEADS local anesthetic inmice pain model.

Methods

LMVV-BUP Preparation

The following two formulation of BUP loaded LMVV were prepared:

10 ml LMVV-BUP

100 ml LMVV-BUP

Ethanolic Lipids Solution

The lipids mixture as powder: HSPC-100/C16-SPM/CHOLESTEROL 3/3/4 moleratio were dissolved in ethanol at 65 C and bath sonication at 65 C for5 min.

TABLE 15 LMVV formulations Ethanol volume ml HSPC-100 gr C16-SPM grCHOLESTEROL gr  1 (in 15 ml vile) 0.225 0.225 0.154 10 (in 50 ml vile)2.25 2.25 1.54

Lipid Hydration

The ethanol lipids solution was added to AS solution at 65 C and mixedfor 1 hour to obtain MLV.

TABLE 16 AS solution preparations AS volume ml AS 285 mOs mg mixing  10(in 50 ml vile)  167 (127 Mm) Shaking incubator 100 (in Erlenmeyer 250ml) 1670 Magnetic stirring under water bath

LMVV Preparation

The MLV were exposed to 10 cycles of freezing in liquid nitrogen (2min/cycle) and thawing in 65 C water bath (about 5 min/cycle). The MLVwere placed in 15 ml vials.

TABLE 17 MLV ml 15 ml vials 10 2 100 10AS Gradient Creation

Transmembrane AS gradient were created by removal of AS from the extraliposome aqueous phase replacing it with saline.

Two methods were used:

-   -   (i) Centrifugation at condition of 2000 g or 4000 g for 5 min or        10 min at 4 c, the supernatant was removed and the pellet was        washed with saline at 4 c. the washing process was repeated 7        times.    -   (ii) Diafiltration at 4 c using hollow cartridge 500000 NMWC cut        off. (washed the AS with 10 time volume of saline)

TABLE 18 AS Gradient formation LMVV ml centrifugation diafiltration 10 2ml LMVV washed with LMVV diluted with saline ×2 8 ml saline, repeated or×5 washed with 200 ml 7 times. or 500 ml saline 100 — No dilution,washed with 1000 ml saline

Bupivacaine Loading

Bupivacaine was loaded into LMVV using remote loading of performedliposomes having a trans membrane ammoniume sulfate (AS) gradient. Thepellet of the LMVV-AS gradient was mixed with filtrated 5.7% BUP in purewater (285 mOsm, PH=4.1) at the volume ratio 1:4 or 1:3 during 1 hour at65 C. The mixture was then cooled to 4 C during 1 hour and thesupernatant was removed from LMVV then saline was added.

TABLE 19 Pellet of AS grad LMVV vials 5.7% BUP ml 10 50 ml 30 ml 2 × 5 2 × 15 ml  2 × 20 ml 10 × 10 10 × 50 ml 10 × 30 ml

Non-encapsulated bup was removed from LMVV by using two methods:

(i) diafiltration

(ii) washing with saline by centrifugation: this procedure was used forthe 100 ml LMVV preparation.

LMVV-BUP pellet 90 ml were washed with 180 ml saline followed by 10 mincentrifugation 4000 g at 4 C (using 9 of 50 ml vials). The supernatantwas replaced with saline 7 times. In the last washing the salinereplaced with 10 mM citrate buffer PH=5.5 in sodium chloride (the finalosmotic pressure 285 mOsm) at a volume ratio 2/1 LMVV pellet/buffer.

TABLE 20 LMVV-BUP Dilution with saline replaced saline volume 10 ×5 500ml 10 ×2 250 mlPreparation of Alginate Beads Entrapping BUP Loaded LMVV

Alginate beads were prepared by Ca++ cross-linked alginate hydro-gelcontaining LMVV-BUP. For sterilization a solution of 2% (w/v) sodiumalginate (ALG) was filtered through 0.2u filter. LMVV-BUP solution wascentrifuged 2000 g for 5 min at 4 C and only the pellet (55.5 mMphospholipids) were used for the beads preparation. Cold 2% (w/v) sodiumalginate solution and cold LMVV-BUP pellet (55.5 mM phos.) 1:1 V/V (0.2ml ALG and 0.2 ml LMVV-BUP) mixture were dipped through a 1 ml syringeinto 15 ml solution of cold 1.54% (w/v) (285 mOsm) calcium chloride.14-17 beads were formed at 10 min gentle mechanical stirring. The beadswere washed with cold saline (10 ml×3) than stored with 400 ul citratebuffer PH=5.5 solution in saline or 0.2%; 0.5%; 2% bup (adjustingosmotic pressure with sodium chloride to get 285 mOsm) at cold room (4C).

Assays

Stability was determined from drug release rate. This was determinedfrom the change level of free drug solution and drug to phospholipidsmolar ratio (D/PL) in the alginate beads at temperature 4 C, 25 C, and37 C.

Characterization of the Beads:

Sample of beads made of 0.2 ml LMVV (pellet) and 0.2 ml 2% alginatewithout storing medium was injected through a #31 needle and the weightof the “broken” beads was measured. This “broken” beads were diluted×10(w/v) with saline and free bup, total bup and total phospholipidsconcentration were determined. Bup was measured by HPLC (Grant et al.Parm. Res 2001 18, 336-343). Phospholipids concentration was determinedby modified Bartlett method (Shmeeda et al 2003 Method Enz. 367,272-292.

Free Bup (Non Liposomal Bup) Determination:

Aliquot of upper phase (sup) from the diluted “broken” beads after 5 min2000 g centrifugation at 4 C followed by adding ×10 volume isopropanol(IPA) to extract the bup for HPLC determination.

Total Bup Determination:

-   -   (I) Total: aliquot of diluted “broken” beads were heated under        boil water 5 min followed by extracting the bup with ×10 volume        in IPA for HPLC determination.    -   (II) Total sup: aliquot of diluted ‘broken” beads were heated        under boil water 5 min and spin it, then aliquot from the upper        phase (sup) was extracted with ×10 volume IPA for HPLC        determination.        Total Phospholipids Determination:

Aliquot of 25 ul or 50 ul from diluted “broken” beads were used forBartlett assay.

Results

Drug Release from LMVV and Beads-LMVV

Bupivacaine release from few LMVV preparations and ALG-beads wasmeasured for different storing solution, as indicated in Table 21.

TABLE 21 LIPOSOME LMVV-BUP PREPARETION STORING SOLUTIONS AND CONDITIONSSterile Volume Storing solution PH conditions preparation Drug/PLSaline, 2% 0.5% bup No buffer − 10 ml 1.5 Saline, 2% 0.5% bup No buffer− 10 ml 2.4 Saline, 2% 0.5% bup 5.5 + 10 ml 1.45 Saline, 2% 0.5% 5.5 +10 ml 1.44 0.2% bup Saline, 2% 1% 5.5 + 100 ml  1.63 0.5% 0.2% bup

TABLE 22 ALGINATE LMVV-BUP BEADS PREPARATION Storing solution Storing pHSterile condition Saline, 2% 0.5% 0.2% bup No buffer − 2% 0.5% bup Nobuffer − Saline, 2% 0.5% bup 5.5 + Saline, 2% 0.5% 0.2% bup 5.5 + 2% bup5.5 + 0.5% bup 5.5 + 0.2% bup 5.5 + saline 5.5 + 1% 5.5 +

FIG. 9A-9H show the release profile of Bupivacaine from alginate LMVVbeads at 4° C. in different storing solution.

Bupivacaine Release from Alginate LMVV Beads at 37° C. and 25° C. inDifferent Storing Solution.

FIGS. 10A and 10B show release of BUP form beads (080209) washed fromthe storing (1 month) solution with saline and incubated at 37 C. FIG.10C shows release of Bup from beads (180209) unwashed from the storing(1 month at 4° C.) solution and then incubated at 37° C. FIG. 10D showsrelease of BUP from beads (010609) unwashed from the storing (1 week at4° C.) solution and then incubated at 37° C. and 25° C.

Characterization the Beads:

TABLE 23 B-ALG Aug. 02, 2009 (IMVV 29 Jan. 2009 D/L = 1.5) After 3months at 4 C. beads sample weight free bup (%) total bup (%) phos (nM)D/L bup in the sample storing in sal 0.302 0.13(0.1) 0.91 (sup)  30.20.99 2.73 after washing  1.29 1.4 3.9 storing in 0.5 0.256 0.23 1.6 30.91.7 4.1 after washing storing in 2% 0.292 0.42 1.25 (SUP) 25.85 1.593.65 after washing 1.8 2.3 5.25 B-ALG Aug. 02, 2009 (IMVV Jan. 06, 2009D/L = 1.63) Beads D after 1 week at 4 c. samples free total sup phostotal sup storing in weight gr bup (%) total bup (%) total bup (%) mMD/L(total) D/L (sup) bup (mg)/sample saline 0.2884 0.12 1.15 1.00 23.201.56 1.35 3.32 2.88 0.2884 0.82 1.00 25.50 1.06 1.24 2.36 2.88 0.2% bup0.1906 0.22 1.29 1.21 25.27 1.60 1.50 2.46 2.31 washed 0.1906 1.28 1.0824.50 1.70 1.44 2.44 2.06 0.5% bup 0.2942 0.33 1.31 1.28 23.00 1.78 1.753.85 3.77 unwashed 0.2942 1.28 0.92 26.25 1.58 1.14 3.77 2.71 0.5% bup0.4037 0.20 1.68 1.46 17.37 1.51 1.31 6.78 5.89 washed 0.4037 1.02 0.7214.18 2.30 1.64 4.12 2.91  2% bup 0.2896 0.38 2.09 1.42 24.53 2.67 1.806.05 4.11 washed 0.2896 1.58 1.34 28.00 1.82 1.56 4.58 3.88 Blank (no)0.1871 32?   bup 25.9  LMVV Jan. 06, 2009 D/L = 1.63) Characterizationof beads B, 2 weeks incubation at 37 C. and washed from the storingsolution storing free total sup Amount of bup (mg)/ solution weight grbup total bup total bup phos mM D/L(total) D/L (sup) sample (total)(sup) 0.5% bup(B) 0.2604 0.19 0.49 0.27 24.8 0.64 0.35 1.27596 0.703080.5% bup(C) 0.1891 0.16 0.38 0.23 20.9 0.6 0.35 0.71858 0.43493  2% bup(B) 0.2457 0.35 0.56 0.41 19.8 0.92 0.68 1.37592 1.00737  2% bup (C)0.2535 0.48 0.61 0.52 20.1 0.98 0.84 1.54635 1.3182 B-ALG Jul. 4, 2009(LMVVFeb. 4, 2009 D/L = 1.45) After 1 month at 4 C. Beads sample Eweight free pup (%) total bup (%) phos (mM) D/L Total bup in the samplestoring in saline 0.39 0.116 0.86 (sup)  25.7 1.09 3.35 unwashed 1.161.48 4.518 storing in 0.5% bup 0.227 0.38 1.1 (sup) 31.6 1.12 2.31unwashed 1.24 1.29 2.6 storing in 2% bup 0.3435 0.51 1.2 (sup) 24.7 1.63.85 after washing 1.5  2 4.81 B-ALG Aug. 2, 2009 After 6 month at 4 C.sample B weight free pup (%) total bup (%) phos (mM) D/L In salineunwashed 0.259 0.1634 1.12 30.12 washed 0.017 0.98 31.5 1.06 in 0.5% bupunwashed 0.2755 0.324 1.36 27.625 washed 0.022 1.04 28 1.21 in 2% bup(washed beads) 0.19 0.096 1.12 26.17 washed after injection 0.018 1.3071.623 LMVV Jan. 06, 2009 D/L = 1.63 Characterization of beads 3 weeksafter incubation at 25 C. and washed (well) from the storing solution:storing free total sup Amount of bup (mg)/ solution weight gr bup totalbup total bup phos mM D/L(total) D/L (sup) sample (total (sup) 0.5% BUP0.1994 0.93 0.74 26.45 1.14 0.92 1.85442 1.47556  2% BUP 0.239 0.0761.26 1.08 17.467 2.35 2.02 3.0114 2.5812Chitosane Beads-LMVV Bup

Bup release from chitosane beads encapsulating Bup loaded LMVV is shownin FIG. 11E.

Analgesic Efficacy in Mouse Model

Testing for analgesia was performed by electrical stimulation of miceskin as described elsewhere (G. J. Grant et al, Pharmaceutical Research,18, no 3, 336-343, 2001). Pain by electrical stimulation at the desiredintensity was applied to the skin of shaved mice abdomen. The currentgenerator (model S48, Grass Instruments (W. Warwick, R.I. USA) was used.Mice (male Swiss-Webster, 26±3 gr (n=8 per group) were used. The micewere shaved the hair overlying the abdomen and tested prior to injectionto determine the Individual vocalization threshold of each mice. Thanthe mice were injected through 30G needle with 0.25 ml of one of thefollowing formulation:

-   -   1—Alginate beads containing LMVV-BUP (010609), were stored at 4        c for 2 months in 0.5% bup, PH=5.5 (see B3). The external        solution of the beads were removed before injection without        washing them.    -   2—Alginate beads containing LMVV-BUP (010609), were stored at 4        c for 2 months in 2% bup, PH=5.5 (see B3). The external solution        of the beads were removed and the beads washed (2.5 ml saline×5)        before injection.    -   3—Alginate beads containing LMVV-BUP (010609), were stored at 4        c for 2 months in 1% bup, PH=5.5 (see B3). The external solution        of the beads were removed before injection without washing them.    -   4—Alginate beads containing LMVV-BUP (010609), were stored at 4        c for 2 months in saline, PH=5.5 (see B3). The external solution        of the beads were removed before injection without washing them.    -   5—LMVV-BUP (010609) were stored at 4 c for 2 months in saline        PH=5.5.    -   6—BLANK: Aginate beads containing LMVV without bup, were stored        at 4 c for 2 months in saline PH=5.5. The external solution of        the beads were removed before injection.    -   7—FREE BUP: 0.15 ml of the clinical 0.5% BUP were injected.

The analgesia duration was than followed as specified in the experimentitself. All experiments were approved and ratified by the HUJI ethiccommittee.

FIG. 12 shows the analgesia duration in mice of each of the above notedformulations.

TABLE 24 FREE BUP (%) in the preparation formulations: FREE BUP(%) FREEBUP(%) SAMPLE time = 0 time time = 2 months FORMULATION of preparationTime of the in-vivo exp. No 1: beads stored in 0.5% 0.33 0.36 bup No 2;beads stored in 2% 0.38 0.5 bup (washed) No 3: beads stored in 1% 0.550.5 bup No 4: beads stored in 0.12 0.192 saline No 5: LMVV 010609 0.0240.038

TABLE 25 BUP DOSE PER MOUSE SAMPLE FREE LIPOSOMAL TOTAL FORMULATION BUP(mg) BUP (mg) BUP (mg) No 1: beads stored in 0.5% 0.96 2.34 3.3 bup No2; beads stored in 2% 1.13 1.82 2.95 bup (washed) No 3: beads stored in1% 0.72 1.79 2.51 bup No 4: beads stored in 0.6 2.0 2.6 saline No 5:LMVV 010609 0.684 3.06 3.75

The invention claimed is:
 1. A composition, comprising: 1) an externalstorage solution having a first osmolality, comprising: at least onewater soluble, lipid permeable active agent having a firstconcentration, wherein the at least one water soluble, lipid permeableactive agent has a molecular weight of less than or equal to 500 Da, anda LogP of greater than 2.2, wherein the at least one water soluble,lipid permeable active agent has a molecular weight of less than orequal to 500 Da, and a LogP of greater than 2.2 is selected from thegroup consisting of chloroprocaine, ciprocaine, cocaine,cyclomethycaine, dimethocaine, etidocaine, propoxycaine, proparacaine,tetracaine, bupivacaine, cinchocaine, levobupivacaine, lidocaine,piperocaine, ropivacaine, dibucaine, heptacaine, mesocaine,propanocaine, carbisocaine, butacaine, betamethasone hemisuccinate andmethyl prednisolone succinate; and 2) particles contained within theexternal storage solution, wherein the particles comprise: i) a waterinsoluble, water absorbed cross-linked polymeric matrix; ii) liposomescontained within the water insoluble, water absorbed cross-linkedpolymeric matrix, wherein the liposomes comprise cholesterol and aphosphatidylcholine lipid having a Tm greater than or equal to 41° C.;wherein the liposomes have an intraliposomal aqueous compartment,containing an intraliposomal solution having a second osmolality,comprising the at least one water soluble, lipid permeable active agenthaving a molecular weight of less than or equal to 500 Da, and a LogP ofgreater than 2.2, at a second concentration; wherein the firstosmolality is not more than 50 mOsmole higher than the secondosmolality; wherein the second concentration is at least three timesgreater than the first concentration; and wherein the firstconcentration increases not more than 2 fold during storage at 4° C. fora period of at least three months.
 2. The composition of claim 1,wherein the liposomes are at least one of multilamellar vesicles (MLVs)or multivesicular vesicles (MVVs).
 3. The composition of claim 1,wherein the polymer of the matrix is a fully cross-linked polymer orpartially cross-linked polymer.
 4. The composition of claim 3, whereinthe polymeric matrix is a hydrogel, the hydrogel comprises at least onecross-linked polysaccharide, the polysaccharide being selected from thegroup consisting of alginate and chitosan.
 5. The composition of claim1, wherein the first concentration of the at least one water soluble,lipid permeable active agent having a molecular weight of less than orequal to 500 Da, and a LogP of greater than 2.2 is between 0.2% to 2.0%.6. The composition of claim 1, wherein the liposomes are largemultivesicular vesicles (LMVV) having a diameter of between 200 nm to 25μm.
 7. The composition of claim 1, wherein the particles areadministered to a subject in need thereof.